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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
  • C12N15/8205—Agrobacterium mediated transformation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
  • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers

Definitions

• the present invention relates to methods and compositions for the transformation of duckweed, particularly to methods for transformation utilizing ballistic bombardment and Agrobacterium.
• the duckweeds are the sole members of the monocotyledonous family, Lemnaceae.
• the four genera and 34 species are all small, free-floating, fresh-water plants whose geographical range spans the entire globe.
• Geobatanischen Institut ETH, founded Rubel, Zurich (1986) Although the most morphologically reduced plants known, most duckweed species have all the tissues and organs of much larger plants, including roots, stems, flowers, seeds and fronds.
• Duckweed species have been studied extensively and a substantial literature exists detailing their ecology, systematics, life-cycle, metabolism, disease and pest susceptibility, their reproductive biology, genetic structure, and cell biology.
• the growth habit of the duckweeds is ideal for microbial culturing methods.
• the plant rapidly proliferates through vegetative budding of new fronds, in a macroscopic manner analogous to asexual propagation in yeast.
• Duckweed proliferates by vegetative budding from m Xeristematic cells.
• the meristematic region is small and is found on the ventral surface of the frond.
• Meristematic cells lie in two pockets, one on each side of the frond midvein.
• the small midvein region is also the site from which the root originates and the stem arises that connects each frond to its mother frond.
• the meristematic pocket is protected by a tissue flap. Fronds bud alternately from these pockets.
• Duckweeds The family of Lemnaceae - A Monograph Study. Geobatanischen Institut ETH, founded Rubel. Zurich (1986)). Ploidy levels are estimated to range from 2-12 C. Id. Genetic diversity within the Lemnaceae has been investigated using secondary products, isozymes , and DN A sequences. McClure and Alston, Nature 4916, 311 (1964); McClure and Alston, Amer. J. Bot. 53, 849 (1966); Vasseur et al., PI. Syst. Evol. Ill, 139 (1991); Crawford and Landolt, Syst. Bot. 10, 389 (1993).
• the present invention is drawn to methods and compositions for the efficient transformation of duckweed.
• the methods involve the use of ballistic bombardment, Agrobacterium, or electroporation to stably introduce and express a nucleotide sequence of interest in transformed duckweed plants.
• any gene(s) or nucleic acid(s) of interest can be introduced into the duckweed plant.
• Transformed duckweed cells, tissues, plants and seed are also provided.
• the present invention provides a method for transforming duckweed with a nucleotide sequence of interest, wherein said nucleotide sequence comprises at least an expression cassette containing a gene which confers resistance to a selection agent, the method comprising the steps of: (a) providing a duckweed tissue target, the cells of the duckweed tissue including cell walls; and (b) propelling the nucleotide sequence at the duckweed tissue target at a velocity sufficient to pierce the cell walls and deposit the nucleotide sequence within a cell of the tissue to thereby produce a transformed tissue, wherein the nucleotide sequence is carried by a microprojectile; and wherein the nucleotide sequence is propelled at the tissue by propelling the microprojectile at the tissue.
• the present invention provides a method for transforming duckweed with a nucleotide sequence of interest, the method comprising the steps of: (a) inoculating a duckweed plant tissue with an Agrobacterium comprising a vector which comprises the nucleotide sequence, wherein the nucleotide sequence comprises at least an expression cassette containing a gene which confers resistance to a selection agent; and (b) co-cultivating the -f tissue with the Agrobacterium to produce transformed tissue.
• the present invention provides a method of transforming duckweed by electroporation.
• the present invention provides transformed duckweed plants and transformed duckweed tissue culture produced by the methods described above.
• the present invention provides a transformed duckweed plant and methods of using transformed duckweed plants to produce a recombinant protein or peptide.
• Duckweed offers an ideal plant-based gene expression system.
• a duckweed gene expression system provides the pivotal technology that would be useful for a number of research and commercial applications.
• a differentiated plant system which can be manipulated with the laboratory convenience of yeast provides a very fast system in which to analyze the developmental and physiological roles of isolated genes.
• Model plants such as tobacco and Arabidopsis are currently used for this purpose by plant molecular biologists. These plants require greenhouse or field facilities for growth (often difficult for plant molecular biologists to obtain).
• Alternative gene expression systems are based on microbial or cell cultures where tissue and developmentally regulated gene expression effects are lost. Heterologous gene expression systems also require restructuring of the gene of interest prior to insertion, an expensive and time- consuming process.
• a duckweed system overcomes both of these problems and is far easier to grow and maintain in a laboratory setting. If it is desirable to harvest the expressed proteins or peptides (or molecules produced thereby), this can be accomplished by any suitable technique known in the art, such as mechanical grinding or lysing of cells.
• a duckweed-based system For commercial production of valuable proteins, a duckweed-based system has a number of advantages over existing microbial or cell culture systems.
• plants show post-translational processing that is similar to mammalian cells, overcoming one major problem associated with microbial cell production of mammalian proteins.
• Duckweed is also far cheaper to produce than mammalian cell cultures. It has already been shown by others (Hiatt, Nature 334, 469 (1990)) that plant systems have the ability to assemble multi-subunit proteins, an ability often lacking in microbial systems r. Plant production of therapeutic proteins also limits the risk from contaminating substances, including animal viruses, produced in mammalian cell cultures and in microbial systems. Contaminating substances are a major concern in therapeutic protein production.
• duckweed can be grown in fermentor/bioreactor vessels, making the system's integration into existing protein production industrial infrastructure far easier.
• duckweed offers the advantage that production is readily scaleable to almost any quantity because it can be grown under field conditions using nutrient-rich wastewater.
• a genetically engineered duckweed system growing on wastewater could produce a valuable product while simultaneously cleaning up wastewater for reuse.
• Such a system would turn a net capital loss (remediation of wastewater from discharge) into a chemical or enzyme production system with a positive economic balance.
• Duckweeds' advantage over chemical syntheses in field crops is that production does not require arable crop land or irrigation water necessary to increase food production for the world's increasing population.
• Figure 1 presents an autoradiograph produced by Southern hybridization of untransformed duckweed DNA and transformed duckweed DNA from line D with a radioactively labeled 3.2 kb fragment from pBI121 containing the GUS gene.
• Channels 1) Isolated, undigested pBI121 DNA. The expected major band is at 12.8 kb. The lower molecular weight band is probably represents the supercoiled plasmid. 2) Hindl ⁇ l digested, pBI121 DNA. This digestion linearizes the plasmid and shows the expected 12.8 kb band. The lower molecular weight band indicates incomplete digestion.
• the present invention is directed to methods for transforming duckweed.
• the methods utilize ballistic bombardment or Agrobacterium to stably transform the duckweed cells.
• the methods use electroporation to transform duckweed.
• the methods and transformed plants of the present invention find use as a plant-based gene expression system possessing many of the advantages of yeast.
• the present invention utilizes one of two systems to stably transform duckweed: ballistic transformation using microprojectile bombardment or Agrobacterium-medis ⁇ ed transformation.
• duckweeds would be expected to be refractory to Agrobacterium transformation because they are monocotyledonous plants, it has unexpectedly been found that duckweed can be transformed using Agrobacterium.
• Transformed duckweed plants according to the present invention may also be generated by electroporation. See, e.g., Dekeyser et al., Plant Cell 2, 591 (1990); D'Halluin et al, Plant Cell 4, 1495 (1992); U.S. Patent No. 5,712,135 to c 1
• One advantage of electroporation is that large pieces of DNA. including artificial chromosomes, can be transformed into duckweed by this method.
• Any suitable duckweed cell or tissue type can be transformed according to the present invention.
• nucleic acids can be introduced into duckweed cells in tissue culture.
• the small size and aquatic growth habit of duckweed plants allows for nucleic acids to be introduced into duckweed cells of intact embryos, fronds, roots, and other organized tissues, such as meristematic tissue.
• nucleic acids can be introduced into duckweed callus.
• transformed duckweed plants produced by the claimed methods exhibit normal morphology and are fertile by sexual reproduction.
• transformed plants of the present invention contain a single copy of the transferred nucleic acid, and the transferred nucleic acid has no notable rearrangements therein.
• duckweed plants in which the transferred nucleic acid is present in low copy numbers i.e., no more than five copies, alternately, no more than three copies, as a further alternative, fewer than three copies of the nucleic acid per transformed cell).
• duckweed refers to members of the family Lemnaceae. There are known four genera and 34 species of duckweed as follows: genus Lemna (L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana); genus Spirodela (S. intermedia, S. polyrrhiza, S. punctata); genus Wolffia (Wa. angusta, Wa. arrhiza, Wa.
• genus Lemna L. aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L. perpusilla, L
• Lemna gibba, Lemna minor, and Lemna miniscula are preferred, with Lemna minor and Lemna miniscula being most preferred.
• Lemna species can be classified using the taxonomic scheme described by Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae - A Monograph Study. Geobatanischen Institut ETH, Stainless Rubel, Zurich (1986)).
• any nucleic acid of interest can be used in the methods of the invention.
• a duckweed plant can be engineered to express disease and insect resistance genes, genes conferring nutritional value, antifungal, antibacterial, or antiviral genes, and the like.
• therapeutic e.g., for veterinary or medical uses
• immunogenic e.g., for vaccination
• peptides and proteins can be expressed using transformed duckweed according to the present invention.
• the method can be used to transfer any nucleic acid for controlling gene expression.
• the nucleic acid to be transferred can encode an antisense oligonucleotide.
• duckweed can be transformed with one or more genes to reproduce enzymatic pathways for chemical synthesis (e.g., for the synthesis of plastics) or other industrial processes (e.g., keratinase).
• the nucleic acid may be from duckweed or from another organism (i.e., heterologous).
• nucleic acids of interest can be obtained from prokaryotes or eukaryotes (e.g., bacteria, fungi, yeast, viruses, plants, mammals) or the nucleic acid sequence can be synthesized in whole or in part.
• the nucleic acid encodes a secreted protein or peptide.
• the transferred nucleic acid to be expressed in the transformed duckweed encodes a protein hormone, growth factor, or cytokine, more preferably, insulin, growth hormone (in particular, human growth hormone), and -interferon.
• the nucleic acid expresses ⁇ -glucocerebrosidase.
• nucleic acids that encode peptides or proteins that cannot effectively be commercially-produced by existing gene expression systems, because of cost or logistical constraints, or both. For example, some proteins cannot be expressed in mammalian systems because the protein interferes with cell viability, cell proliferation, cellular differentiation, or protein assembly in mammalian cells.
• Such proteins include, but are not limited to, retinoblastoma protein, p53, angiostatin and leptin.
• the present invention can be advantageously employed to produce mammalian regulatory proteins; it is unlikely given the large evolutionary distance between higher plants and mammals that these proteins will interfere with regulatory processes in duckweed.
• Transgenic duckweed can also be used to produce large quantities of proteins such as serum albumin (in particular, human serum albumin), hemoglobin and collagen, which challenge the production capabilities of existing expression systems.
• biologically-active multimeric proteins e.g., monoclonal antibodies, hemoglobin, P450 oxidase, and collagen, and the like
• biologically-active multimeric proteins e.g., monoclonal antibodies, hemoglobin, P450 oxidase, and collagen, and the like
• biologically active includes multimeric proteins in which the biological activity is altered as compared with the native protein (e.g, suppressed or enhanced), as long as the protein has sufficient activity to be of interest for use in industrial or chemical processes or as a therapeutic, vaccine, or diagnostics reagent.
• One exemplary approach for producing biologically-active multimeric proteins in duckweed uses an expression vector containing the genes encoding all of the polypeptide subunits. See, e.g., During et al. (1990) Plant Molecular Biology 15:281 ; van Engelen et al, (1994) Plant Molecular Biology 26:1701. This vector is then introduced into duckweed cells using any known transformation method, such as a gene gun or Agrobacterium-mediated transformation. This method results in clonal cell lines that express all of the polypeptides necessary to assemble the multimeric protein. As one alternate method, independent vector constructs are made that encode each polypeptide subunit.
• Each of these vectors is used to generate separate clonal lines of transgenic plants expressing only one of the necessary polypeptides. These transgenic plants are then crossed to create progeny that express all of the necessary polypeptides within a single plant. See Hiatt et al., (1989) Nature 342:76; U.S. Patent Nos. 5,202,422 and 5,639,947 to Hiatt et al.
• a variation on this approach is to make single gene constructs, mix DNA from these constructs together, then deliver this mixture of DNAs into plant cells using ballistic bombardment or Agrobacterium- mediated transformation, more preferably ballistic bombardment.
• some or all of the vectors may encode more than one subunit of the multimeric protein (i.e., so that there are fewer duckweed clones to be crossed than the number of subunits in the multimeric protein).
• the nucleic acid to be transferred is contained within an expression cassette.
• the expression cassette comprises a transcriptional initiation region linked to the nucleic acid or gene of interest.
• Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene or genes of interest (e.g., one gene of interest, two genes of interest, etc.) to be under the transcriptional regulation of the regulatory regions.
• the expression cassette encodes a single gene of interest.
• the nucleic acid to be transferred contains two or more expression cassettes, each of which encodes at least one gene of interest (preferably one gene of interest).
• the transcriptional initiation region (e.g., a promoter) may be native or homologous or foreign or heterologous to the host, or could be the natural sequence or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.
• a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
• Any suitable promoter known in the art can be employed according to the present invention (including bacterial, yeast, fungal, insect, mammalian, and plant promoters). Plant promoters are preferred, with duckweed promoters being most preferred. Exemplary promoters include, but are not limited to, the Cauliflower Mosaic Virus 35S promoter, the opine synthetase promoters (e.g., nos, mas, ocs, etc.), the ubiquitin promoter, the actin promoter, the ribulose bisphosphate (RubP) carboxylase small subunit promoter, and the alcohol dehydrogenase promoter.
• the duckweed RubP carboxylase small subunit promoter is known in the art.
• promoters from viruses that infect plants, preferably duckweed are also suitable including, but not limited to, promoters isolated from Dasheen mosaic virus, Chlorella virus (e.g., the Chlorella virus adenine methyltransferase promoter; Mitra et al., (1994) Plant Molecular Biology 26:85), tomato spotted wilt virus, tobacco rattle virus, tobacco necrosis virus, tobacco ring spot virus, tomato ring spot virus, cucumber mosaic virus, peanut stump virus, alfalfa mosaic virus, and the like. //
• promoters can be chosen to give a desired level of regulation.
• a promoter that confers constitutive expression e.g, the ubiquitin promoter, the RubP carboxylase gene family promoters, and the actin gene family promoters.
• promoters that are activated in response to specific environmental stimuli (e.g., heat shock gene promoters, drought-inducible gene promoters, pathogen-inducible gene promoters, wound-inducible gene promoters, and light/dark-inducible gene promoters) or plant growth regulators (e.g., promoters from genes induced by abscissic acid, auxins, cytokinins, and gibberellic acid).
• promoters can be chosen that give tissue-specific expression (e.g., root, leaf and floral-specific promoters).
• the transcriptional cassette includes in the 5 ' - 3 ' direction of transcription, a transcriptional and translational initiation region, a nucleotide sequence of interest, and a transcriptional and translational termination region functional in plants.
• Any suitable termination sequence known in the art may be used in accordance with the present invention.
• the termination region may be native with the transcriptional initiation region, may be native with the nucleotide sequence of interest, or may be derived from another source.
• Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthetase and nopaline synthetase termination regions. See also, Guerineau et al., Mol. Gen. Genet.
• the gene(s) of interest can be provided on any other suitable expression cassette known in the art.
• the gene(s) may be optimized for increased expression in the transformed plant.
• mammalian, yeast or bacterial or plant dicot genes are used in the invention, they can be synthesized using monocot or duckweed preferred codons for improved expression. Methods are available in the art for synthesizing plant preferred genes. See, e.g., United States Patent Nos. 5,380,831 ; 5,4 I3t6,391 ; and Murray et al., Nucleic Acids. Res. 17, 477 (1989); herein incorporated by reference.
• the expression cassettes may additionally contain 5' leader sequences.
• leader sequences can act to enhance translation.
• Translation leaders are known in the art and include: picornavirus leaders, e.g., EMCV leader (Encephalomyocarditis 5 ' noncoding region; Elroy-Stein et al., Proc. Natl. Acad.
• potyvirus leaders e.g., TEV leader (Tobacco Etch Virus; Allison et al., Virology, 154, 9 (1986)); human immunoglobulin heavy-chain binding protein (BiP; Macajak and Sarnow, Nature 353, 90 (1991)); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4; Jobling and Gehrke, Nature 325, 622 (1987)); tobacco mosaic virus leader (TMV; Gallie, MOLECULAR BIOLOGY OF RNA, 237-56 (1989)); and maize chlorotic mottle virus leader (MCMV; Lommel et al., Virology 81, 382 (1991)). See also, Della-Cioppa et al., Plant Physiology 84, 965 (1987). Other methods known to enhance translation can also be utilized, e.g., introns and the like.
• the exogenous nucleic acid of interest may additionally be operably associated with a nucleic acid sequence that encodes a transit peptide that directs expression of the encoded peptide or protein of interest to a particular cellular compartment.
• Transit peptides that target protein accumulation in higher plants cells to the chloroplast, mitochondrion, vacuole, nucleus, and the endoplasmic reticulum (for secretion outside of the cell) are known in the art.
• the transit peptide targets the protein expressed from the exogenous nucleic acid to the chloroplast or the endoplasmic reticulum. Transit peptides that target proteins to the endoplasmic reticulum are desirable for correct processing of secreted proteins.
• Targeting protein expression to the chloroplast has been shown to result in the accumulation of very high concentrations of recombinant protein in this organelle.
• a duckweed nucleic acid encoding an RubP carboxylase transit peptide has already been cloned. Stiekma et al., (1983) Nucl. Acids Res. 11:8051-61; see also U.S. Patent Nos. 5,717,084 and 5,728,925 to Herrera-Estrella et al.
• the pea RubP carboxylase small subunit transit peptide sequence has been used to express and target mammalian genes in plants.
• mammalian transit peptides can be used to target recombinant protein expression, for example, to the mitochondrion and endoplasmic reticulum. It has been demonstrated that plant cells recognize mammalian transit peptides that target endoplasmic reticulum.
• the expression cassettes may contain more than one gene or nucleic acid sequence to be transferred and expressed in the transformed plant. Thus, each nucleic acid sequence will be operably linked to 5 ' and 3' regulatory sequences. Alternatively, multiple expression cassettes may be provided.
• the expression cassette will comprise a selectable marker gene for the selection of transformed cells.
• Selectable marker genes are utilized for the selection of transformed cells or tissues.
• Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds.
• Herbicide resistance genes generally code for a modified target protein insensitive to the herbicide or for an enzyme that degrades or detoxifies the herbicide in the plant before it can act. See, DeBlock et al., EMBO J. 6, 2513 (1987); DeBlock et al., Plant Physiol.
• EPSPS 5-enolpyruvylshikimate-3- phosphate synthase
• ALS acetolactate synthase
• selectable marker genes include, but are not limited to, genes encoding: neomycin phosphotransferase II (Fraley et al., CRC Critical Reviews in Plant Science 4, 1 (1986)); cyanamide hydratase (Maier-Greiner et al., Proc. Natl. Acad. Sci. USA 88, 4250 (1991)); aspartate kinase; dihydrodipicolinate synthase (Perl et al., BioTechnology 11, 715 (1993)); bar gene (Toki et al., Plant Physiol. 100, 1503 (1992); Meagher et al., Crop Sci.
• the bar gene confers herbicide resistance to glufosinate-type herbicides, such as phosphinothricin (PPT) or bialaphos, and the like.
• PPT phosphinothricin
• other selectable markers that could be used in the vector constructs include, but are not limited to, the pat gene, also for bialaphos and phosphinothricin resistance, the ALS gene for imidazolinone resistance, the HPH or HYG gene for hygromycin resistance, the EPSP synthase gene for glyphosate resistance, the Hm ⁇ gene for resistance to the Hc-toxin, and other selective agents used routinely and known to one of ordinary skill in the art. See generally, Yarranton, Curr. Opin. Biotech.
• selectable marker genes are not meant to be limiting. Any selectable marker gene can be used in the present invention.
• the selectable marker genes and other gene(s) and nucleic acids of interest to be transferred can be synthesized for optimal expression in duckweed. That is, the coding sequence of the genes can be modified to enhance expression in duckweed.
• the synthetic nucleic acid is designed to be expressed in the transformed tissues and plants at a higher level. The use of optimized selectable marker genes may result in higher transformation efficiency.
• the nucleotide sequence can be optimized for expression in duckweed or alternatively can be modified for optimal expression in monocots.
• the plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in duckweed. It is recognized that genes which have been optimized for expression in duckweed and other monocots can be used in the methods of the invention. See, e.g., EP 0 359 472, EP 0 385 962, WO 91/16432; Perlak et al., Proc. Natl. Acad. Sci. USA 88, 3324 (1991), and Murray et al., Nuc. Acids Res. 17, 477 (1989), and the like, herein incorporated by reference. It is further recognized that all or any part of the gene sequence may be optimized or synthetic. In other words, fully optimized or partially optimized sequences may also be used.
• Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences which may be deleterious to gene expression.
• the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell.
• the sequence is modified to avoid predicted hairpin secondary mRNA structures.
• the methods of the invention are useful for transforming duckweed plant cells, preferably frond and meristematic cells.
• Such cells also include callus, which can be originated from any tissue of duckweed plants.
• the tissue utilized in initiating callus is meristematic tissue.
• the callus can be originated from any other frond cells, or in principal from any other duckweed tissue capable of forming callus.
• any tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis may be employed to transform duckweed according to the present invention.
• organogenesis as used herein, means a process whereby fronds and roots are developed sequentially from meristematic centers.
• embryogenesis as used herein, means a process whereby fronds and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.
• the method can also be used to transform cell suspensions.
• cell suspensions can be formed from any duckweed tissue.
• the duckweeds make three kinds of callus: (a) a compact, semi-organized callus (designated Type I); (b) a friable, white, undifferentiated callus (designated Type II); and (c) a green, differentiated callus (designated Type III).
• callus can only regenerate plants two ways: via embryos and via shoot formation (in duckweed the frond is the shoot). Methods of callus induction are known in the art, and the particular conditions to be employed can be optimized for each duckweed species and for the type of callus desired, as demonstrated in the Examples below.
• Type I or Type III callus is used to transform duckweed according to the present invention.
• Callus can be induced by cultivating duckweed tissue in medium containing plant growth regulators, i.e., cytokinins and auxins.
• Preferred auxins for callus induction from duckweed tissue include 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NAA).
• Preferred auxin concentrations are 1-30 ⁇ M, more preferably 5-20 ⁇ M, yet more preferably 5-10 ⁇ M.
• the preferred cytokinin is benzyladenine (BA) or thidiazuron (TDZ).
• Preferred cytokinin concentrations are 0.1-10 ⁇ M, more preferably 0.5-5 ⁇ M, ye nt more preferably 0.5-1 ⁇ M.
• callus is induced by cultivating duckweed tissue in medium containing both BA or TDZ and either 2,4-D or NAA.
• auxin or "weak” auxins e.g., indoleacetic acid
• high concentrations of auxin or "strong” auxins e g. , 2,4- D
• basal media for callus formation include N6 medium (Chu et al., Scientia Sinica 18, 659 (1975)) and Murashige and Skoog medium (Murashige and Skoog, Physiol. Plant.
• callus induction frequency is variable. In these species, callus may not be visible for two to three weeks in culture, and it may take four to eight weeks of cultivation before calli are of sufficient size for transformation. Preferably, callus induction is carried out for a period of 1-10 weeks, more preferably 2-8 weeks, yet more preferably 3-5 weeks.
• the preferred media are as for callus induction, but the auxin concentration is reduced.
• One embodiment of the invention is a method of transforming duckweed with a nucleotide sequence of interest, wherein the nucleotide sequence contains at least an expression cassette carrying a gene that confers resistance to a selection agent.
• the nucleotide sequence is carried by a microprojectile.
• the ballistic transformation method comprises the steps of: (a) providing a duckweed tissue as a target; (b) propelling the microprojectile carrying the nucleotide sequence at the duckweed tissue at a velocity sufficient to pierce the walls of the cells within the tissue and to deposit the nucleotide sequence within a cell of the tissue to thereby provide a transformed tissue.
• the method further includes the step of culturing the transformed tissue with a selection agent, as described below.
• the selection step is followed by the step of regenerating transformed duckweed plants from the transformed tissue.
• the technique could be carried out with the nucleotide sequence as a precip , t a,e ( possiblye trise. f .ee Z , drM ) a,oillere, h p/le of th e felicit solut , carefully coumble taining , h . nucleotide sequence.
• Any ballistic cell transformation apparatus can be used in practicing the present invention.
• Exemplary apparatus are disclosed by Sandford et al. (Paniculate Science and Technology 5, 27 (1988)), Klein et al. (Nature 327, 70 (1987)), and in EP 0 270 356.
• Such apparatus have been used to transform maize cells (Klein et al., Proc. Natl. Acad. Sci. USA 85, 4305 (1988)), soybean callus (Christou et al., Plant Physiol.
• helium gene gun (PDS-1000/ ⁇ e) manufactured by DuPont was employed.
• PDS-1000/ ⁇ e helium gene gun
• an apparatus configured as described by Klein et al. (Nature 70, 327 (1987)) may be utilized.
• This apparatus comprises a bombardment chamber, which is divided into two separate compartments by an adjustable-height stopping plate.
• An acceleration tube is mounted on top of the bombardment chamber.
• a macroprojectile is propelled down the acceleration tube at the stopping plate by a gunpowder charge.
• the stopping plate has a bore hole formed therein, which is smaller in diameter than the microprojectile.
• the macroprojectile carries the microprojectile(s), and the macroprojectile is aimed and fired at the bore hole.
• the microprojectile(s) When the macroprojectile is stopped by the stopping plate, the microprojectile(s) is propelled through the bore hole.
• the target tissue is positioned in the bombardment chamber so that a microprojectile(s) propelled through the bore hole penetrates the cell walls of the cells in the target tissue and deposit the nucleotide sequence of interest carried thereon in the cells of the target tissue.
• the bombardment chamber is partially evacuated prior to use to prevent atmospheric drag from unduly slowing the microprojectiles.
• the chamber is only partially evacuated so that the target tissue is not desiccated during bombardment. A vacuum of between about 400 to about 800 millimeters of mercury is suitable. In alternate embodiments, ballistic transformation is achieved without use of microprojectiles.
• an aqueous solution containing the nucleotide sequence of interest as a precipitate could be carried by the macroprojectile (e.g., by placing the aqueous solution directly on the plate-contact end of the macroprojectile without a microprojectile, where it is held by surface tension), and the solution alone propelled at the plant tissue target (e.g., by propelling the macroprojectile down the acceleration tube in the same manner as described above).
• Other approaches include placing the nucleic acid precipitate itself ("wet" precipitate) or a freeze-dried nucleotide precipitate directly on the plate-contact end of the macroprojectile without a microprojectile.
• nucleotide sequence In the absence of a microprojectile, it is believed that the nucleotide sequence must either be propelled at the tissue target at a greater velocity than that needed if carried by a microprojectile, or the nucleotide sequenced caused to travel a shorter distance to the target tissue (or both).
• the microprojectile may be formed from any material having sufficient density and cohesiveness to be propelled through the cell wall, given the particle's velocity and the distance the particle must travel.
• materials for making microprojectiles include metal, glass, silica, ice, polyethylene, polypropylene, polycarbonate, and carbon compounds (e.g., graphite, diamond).
• Metallic particles are currently preferred.
• suitable metals include tungsten, gold, and iridium.
• the particles should be of a size sufficiently small to avoid excessive disruption of the cells they contact in the target tissue, and sufficiently large to provide the inertia required to penetrate to the cell of interest in the target tissue. Particles ranging in diameter from about one-half micrometer to about three micrometers are suitable. Particles need not be spherical, as surface irregularities on the particles may enhance their DNA carrying capacity.
• the nucleotide sequence may be immobilized on the particle by precipitation.
• the precise precipitation parameters employed will vary depending upon factors such as the particle acceleration procedure employed, as is known in the art.
• the carrier particles may optionally be coated with an encapsulating agents such as polylysine to improve the stability of nucleotide sequences immobilized thereon, as discussed in EP 0 270 356 (column 8).
• transformants may be selected and transformed duckweed plants regenerated as described below in Section E.
• duckweed is transformed using Agrobacterium tumefaciens or Agrobacterium rhizogenes, preferably Agrobacterium tumefaciens.
• Agrobacterium-mediated gene transfer exploits the natural ability of A. tumefaciens and A. rhizogenes to transfer DNA into plant chromosomes.
• Agrobacterium is a plant pathogen that transfers a set of genes encoded in a region called T-DNA of the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, into plant cells.
• the typical result of transfer of the Ti plasmid is a tumorous growth called a crown gall in which the T-DNA is stably integrated into a host chromosome. Integration of the Ri plasmid into the host chromosomal DNA results in a condition known as "hairy root disease".
• hairy root disease The ability to cause disease in the host plant can be removed by deletion of the genes in the T-DNA without loss of DNA transfer and integration.
• the DNA to be transferred is attached to border sequences that define the end points of an integrated T-DNA.
• A. rhizogenes Transformation using A. rhizogenes has developed analogously to that of tumefaciens and has been successfully utilized to transform, for example, alfalfa, Solanum nigrum L., and poplar.
• U.S. Patent No. 5, 773,693 to Burgess et al. it is preferable to use a disarmed A. tumefaciens strain (as described below), however, the wild-type A. rhizogenes may be employed.
• An illustrative strain of A. rhizogenes is strain 15834.
• the Agrobacterium strain utilized in the methods of the present invention is modified to contain a gene or genes of interest, or a nucleic acid to be expressed in the transformed cells.
• the nucleic acid to be transferred is incorporated into the T-region and is flanked by T-DNA border sequences.
• a variety of Agrobacterium strains are known in the art particularly for dicotyledon transformation. Such Agrobacterium can be used in the methods of the invention. See, e.g., Hooykaas, Plant Mol. Biol. 13, 327 (1989); Smith et al., Crop Science 35, 301 (1995); Chilton, Proc. Natl. Acad. Sci. USA 90, 3119 (1993); Mollony et al., Monograph Theor. Appl. Genet NY 19, 148 1
• the Ti (or Ri) plasmid contains a vir region.
• the vir region is important for efficient transformation, and appears to be species-specific.
• Binary vector systems have been developed where the manipulated disarmed T-DNA carrying foreign DNA and the vir functions are present on separate plasmids. In this manner, a modified T-DNA region comprising foreign DNA (the nucleic acid to be transferred) is constructed in a small plasmid which replicates in E. coli.
• This plasmid is transferred conjugatively in a tri-parental mating or via electroporation into A. tumefaciens that contains a compatible plasmid with virulence gene sequences. The vir functions are supplied in trans to transfer the T-DNA into the plant genome. Such binary vectors are useful in the practice of the present invention.
• C58-derived vectors are employed to transform A. tumefaciens.
• super-binary vectors are employed. See, e.g., United States Patent No. 5,591,615 and EP 0 604 662, herein incorporated by reference.
• Such a super-binary vector has been constructed containing a DNA region originating from the hypervirulence region of the Ti plasmid pTiBo542 (Jin et al., J. Bacteriol. 169, 4417 (1987)) contained in a super-virulent A. tumefaciens A281 exhibiting extremely high transformation efficiency (Hood et al., Biotechnol.
• Exemplary super-binary vectors known to those skilled in the art include pTOK162 (Japanese patent Appl. (Kokai) No. 4-222527, EP 504,869, EP 604,662, and United States Patent No. 5,591,616, herein incorporated by reference) and pTOK233 (Komari, Plant Cell Reports 9,303 (1990); Ishida et al., Nature Biotechnology 14, 745 (1996); herein incorporated by reference). Other super-binary vectors may be constructed by the methods set forth in the above references.
• Super- binary vector pTOK162 is capable of replication in both E. coli and in A. tumefaciens.
• the vector contains the v/rB, v/rC and virG genes from the virulence region of pTiBo542.
• the plasmid also contains an antibiotic resistance gene, a selectable marker gene, and the nucleic acid of interest to be transformed into the plant.
• the nucleic acid to be inserted into the duckweed genome is located between the two border sequences of the T region.
• Super-binary vectors of the invention can be constructed having the features described above for pTOK162.
• the T-region of the super-binary vectors and other vectors for use in the invention are constructed to have restriction sites for the insertion of the genes to be delivered.
• the DNA to be transformed can be inserted in the T-DNA region of the vector by utilizing in vivo homologous recombination.
• in vivo homologous recombination See, Herrera-Esterella et al., EMBO J. 2, 987 (1983); Horch et al., Science 223, 496 (1984).
• homologous recombination relies on the fact that the super-binary vector has a region homologous with a region of pBR322 or other similar plasmids.
• a desired gene is inserted into the super-binary vector by genetic recombination via the homologous regions.
• any suitable vector for transforming duckweed may be employed according to the present invention.
• the heterologous nucleic acid sequence of interest and the flanking T-DNA can be carried by a binary vector lacking the vir region. The vir region is then provided on a disarmed Ti- plasmid or on a second binary plasmid.
• the heterologous nucleic acid sequence and the T-DNA border sequences can be put into the T-DNA site on the Ti-plasmid through a double recombination event by which the new T-DNA replaces the original Ti-plasmid T- DNA.
• the vir region can be supplied by the Ti-plasmid or on a binary plasmid.
• heterologous nucleic acid sequence and flanking T-DNA can be integrated into the bacterial chromosome as described by U.S. Patent No. 4,940,838 to Schilperoort et al., and the vir region can then be supplied on a Ti- plasmid or on a binary plasmid.
• the Agrobacterium-mediated transformation process of the present invention can be thought of as comprising several steps.
• the basic steps include an infection step and a co-cultivation step. In some embodiments, these steps are followed by a selection step, and in other embodiments by a selection and a regeneration step.
• An optional pre-culture step may be included prior to the infection step.
• the pre-culture step involves culturing the callus, frond, or other target tissue prior to the infection step on a suitable medium.
• the pre-culture period may vary from about 1 to 21 days, preferably 7 to 14 days.
• Such a pre-culture step was found to prevent transformation of maize cultures. See, e.g., EP 0 672 752.
• the cells to be transformed are exposed to Agrobacterium.
• the cells are brought into contact with the Agrobacterium, typically in a liquid medium.
• the Agrobacterium has been modified to contain a gene or nucleic acid of interest.
• the nucleic acid is inserted into the T-DNA region of the vector.
• General molecular biology techniques used in the invention are well- known by those of skill in the art. See, e.g , SAMBROOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL (1989).
• Agrobacterium containing the plasmid of interest are preferably maintained on
• Agrobacterium master plates with stock frozen at about -80°C. Master plates can be used to inoculate agar plates to obtain Agrobacterium that is then resuspended in medium for use in the infection process. Alternatively, bacteria from the master plate can be used to inoculate broth cultures that are grown to logarithmic phase prior to transformation.
• the concentration of Agrobacterium used in the infection step and co- cultivation step can affect the transformation frequency. Likewise, very high concentrations of Agrobacterium may damage the tissue to be transformed and result in a reduced callus response. Thus, the concentration of Agrobacterium useful in the methods of the invention may vary depending on the Agrobacterium strain utilized, the tissue being transformed, the duckweed species being transformed, and the like. To optimize the transformation protocol for a particular duckweed species or tissue, the tissue to be transformed can be incubated with various concentrations of Agrobacterium. Likewise, the level of marker gene expression and the transformation efficiency can be assessed for various Agrobacterium concentrations.
• the concentration of Agrobacterium may vary, generally a concentration range of about 1 x 10 3 cfu ml to about 1 x 10 10 cfu/ml is employed, preferably within the range of about 1 x 10 3 cfu/ml to about 1 x 10 9 cfu/ml, and still more preferably at aboutl x 10 8 cfu/ml to about 1 x 10 9 cfu/ml will be utilized.
• the tissue to be transformed is generally added to the Agrobacterium suspension in a liquid contact phase containing a concentration of Agrobacterium to optimize transformation efficiencies.
• the contact phase facilitates maximum contact of the tissue to be transformed with the suspension of Agrobacterium.
• Infection is generally allowed to proceed for 1 to 30 minutes, preferably 1 to 20 minutes, more preferably 2 to 10 minutes, yet more preferably 3 to 5 minutes prior to the co- cultivation step.
• the conditions can be optimized to achieve the highest level of infection and transformation by Agrobacterium.
• the cells are subjected to osmotic pressure (e.g., 0.6 M mannitol) during the infection and co-cultivation steps.
• tissue may be cultured in medium containing an auxin, such as IAA, to promote cell proliferation (i.e., it is believed that Agrobacterium integrates into the genome during mitosis).
• tissue wounding, vacuum pressure, or cultivation in medium containing acetosyringone can be employed to promote the transformation efficiency.
• the cells to be transformed are co-cultivated with Agrobacterium.
• the co-cultivation takes place on a solid medium.
• Any suitable medium such as Schenk and Hildebrandt medium (Schenk and Hildebrandt, Can. J. Bot. 50, 199 (1972)) containing 1% sucrose and 0.6% agar, may be utilized.
• the optimal co-cultivation time varies with the particular tissue.
• Fronds are co- cultivated with the Agrobacterium for about 2 to 7 days, preferably 2 to 5 days, more preferably 3 to 5 days, and more preferably 4 days.
• callus is co-cultivated with the Agrobacterium for 0.5 to 4 days, more preferably 1 to 3 days, more preferably 2 days.
• Co-cultivation may be carried out in the dark or under subdued light conditions to enhance the transformation efficiency. Additionally, as described above for the inoculation step, co-culturing can be done on medium containing IAA or acetosyringone to promote transformation efficiency. Finally, the co-culturing step may be performed in the presence of cytokinins, which act to enhance cell proliferation.
• the transformed tissue may be subjected to an optional resting and decontamination step.
• the transformed cells are transferred to a second medium containing an antibiotic capable of inhibiting the growth of Agrobacterium.
• This resting phase is performed in the absence of any selective pressures to permit recovery and proliferation of transformed cells containing the heterologous nucleic acid.
• An antibiotic is added to inhibit Agrobacterium growth.
• antibiotics are known in the art which inhibit Agrobacterium and include cefotaxime, timetin, vancomycin, carbenicillin, and the like. Concentrations of the antibiotic will vary according to what is standard for each antibiotic.
• concentrations of carbenicillin will range from about 50 mg/1 to about 250 mg/1 carbenicillin in solid media, preferably about 75 mg/1 to about 200 mg/1, more preferably about 100-125 mg/1.
• concentrations of carbenicillin can be optimized for a particular transformation protocol without undue experimentation.
• the resting phase cultures are preferably allowed to rest in the dark or under subdued light, preferably in subdued light. Any of the media known in the art can be utilized for the resting step.
• the resting/decontamination step may be carried out for as long as is necessary to inhibit the growth of Agrobacterium and to increase the number of transformed cells prior to selection.
• the resting/decontamination step may be carried out for 1 to 6 weeks, preferably 2 to 4 weeks, more preferably 2 to 3 weeks prior to the selection step.
• the selection period is started within 3 weeks following co-cultivation.
• Some strains of Agrobacterium are more antibiotic resistant than are others.
• decontamination is typically performed by adding fresh decontamination medium to the calli every five days or so.
• a stronger antibiotic e.g., vancomycin
• transformants may be selected and duckweed plants regenerated as described below in Section E.
• Duckweed tissue or callus is transformed according to the present invention, for example by ballistic bombardment or Agrobacterium-mediated transformation, each of which is described in more detail above in Sections C and D, respectively.
• the transformed tissue is exposed to selective pressure to select for those cells that have received and are expressing the polypeptide from the heterologous nucleic acid introduced by the expression cassette.
• the agent used to select for transformants will select for preferential growth of cells containing at least one selectable marker insert positioned within the expression cassette and delivered by ballistic bombardment or by the Agrobacterium.
• the conditions under which selection for transformants are generally the most critical aspect of the methods disclosed herein.
• the transformation process subjects the cells to stress, and the selection process can be toxic even to transformants.
• the transformed tissue is initially subject to weak selection, utilizing low concentrations of the selection agent and subdued light (e.g., 1-5 ⁇ mol/m 2 ⁇ sec, with a gradual increase in the applied selection gradient by increasing the concentration of the selection agent and/or increasing the light intensity.
• Selection pressure may be removed altogether for a time and then reapplied if the tissue looks stressed. Additionally, the transformed tissue may be given a "resting" period, as described above in Section D, before any selection pressure is applied at all.
• the selection medium generally contains a simple carbohydrate, such as 1% to 3% sucrose, so that the cells do not carry out photosynthesis.
• the selection is initially performed under subdued light conditions, or even in complete darkness, so as to keep the metabolic activity of the cells at a relatively low level.
• specific conditions under which selection is performed can be optimized for every species or strain of duckweed and for every tissue type being transformed without undue experimentation.
• selection step There is no particular time limit for the selection step. In general, selection will be carried out long enough to kill non-transformants and to allow transformed cells to proliferate at a similar rate to non-transformed cells in order to generate a sufficient callal mass prior to the regeneration step. Thus, the selection period will be longer with cells that proliferate at a slower rate.
• Type I duckweed callus for example, proliferates relatively slowly and selection may be carried out for 8-10 weeks prior to regeneration. Methods of regenerating certain plants from transformed cells and callus are known in the art. See, e.g., Kamo et al., Bot. Gaz.
• any method known in the art may be utilized to verify that the regenerating plants are, in fact, transformed with the transferred nucleic acid of interest.
• histochemical staining, ELISA assay, Southern hybridization, Northern hybridization, Western hybridization, PCR, and the like can be used to detect the transferred nucleic acids or protein in the callal tissue and regenerating plants.
• duckweed can be transformed utilizing ballistic bombardment and Agrobacterium
• alterations to the general methods described herein can be used to increase efficiency or to transform strains that may exhibit some recalcitrance to transformation.
• Factors that affect the efficiency of transformation include the species of duckweed, the tissue infected, composition of the media for tissue culture, selectable marker genes, the length of any of the above- described step, kinds of vectors, and light/dark conditions.
• concentration and strain of A. tumefaciens or A. rhizogenes must also be considered. Therefore, these and other factors may be varied to determine what is an optimal transformation protocol for any particular duckweed species or strain.
• This section presents experiments pertaining to methods of making duckweed callus.
• a number of examples use Lemna gibba G3 as the duckweed strain, the strain used to optimize culturing parameters: (1) basal medium formulation, (2) type and concentration of plant growth regulators, and (3) transfer schedule.
• the duckweeds make three kinds of callus: (a) a compact, semi-organized callus (designated Type I); (b) a friable, white, undifferentiated callus (designated Type II); and (c) a green, differentiated callus (designated Type III).
• tissue culture callus can only regenerate plants two ways: via embryos and via shoot formation (in duckweed the frond is the shoot). The data presented below demonstrate that transformed duckweed plants can be regenerated from all known pathways of callus regeneration of fronds.
• Example 1 Eighteen combinations of an auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), and a cytokinin, benzyladenine (BA), were tested for their effects on callus induction in a duckweed species, Lemna gibba G3. Duckweed fronds were grown in liquid Hoagland's medium (Hoagland and
• This callus type was produced by greater than 50% of all fronds proliferated during the incubation time. All three types of callus demonstrated proliferation at all 18 2,4-D and B A combinations in varying frequencies. Callus proliferation was the most vigorous in a broad range of 2,4-D concentrations, from 20-50 ⁇ M, and BA concentrations between 0.01 and 0.1 ⁇ M.
• Duckweed fronds were grown in liquid Hoagland's medium containing 3% sucrose for two weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m ⁇ sec. prior to experimentation.
• forty 100ml portions of Murashige and Skoog medium with 3% sucrose, 0.15%) Gelrite, and 0.4% Difco Bacto-agar were prepared with 2,4-D concentrations of 20, 30, 40, 50, 60, 70, 80, 100 ⁇ M and BA concentrations of 0.01, 0.05, 0.1, 0.5, and 1.0 ⁇ M. All media were pH adjusted to 5.8, autoclaved at 121°C for 20 minutes, cooled, and each 100 ml was poured into 4, 100mm x 15 mm petri dishes.
• Duckweed fronds were grown in liquid Hoagland's medium containing 3% sucrose for two weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec. prior to experimentation.
• forty 100ml portions of Murashige and Skoog medium with 3% sucrose, 0.15% Gelrite, and 0.4% Difco Bacto-agar were prepared with dicamba concentrations of 10, 20, 30, 40, 50, 60, 80, 100 ⁇ M and BA concentrations of 0.01 , 0.05, 0.1, 0.5, and 1.0 ⁇ M. All media were pH adjusted to 5.8, autoclaved at 121°C for 20 minutes, cooled, and each 100 ml was poured into 4, 100mm x 15 mm petri dishes.
• Type I (2) Type II, and (3) a Type III callus.
• callus proliferation was poor and occurred on dicamba concentrations of 10 and 20 ⁇ M; above 30 ⁇ M callus proliferation did not occur.
• Type II and Type III callus proliferated in response to dicamba; Type I callus proliferation was rare.
• Duckweed fronds were grown in liquid Schenk and Hildebrandt medium (Schenk and Hildebrandt, Can. J. Bot. 50, 199 (1972)) containing 1% sucrose for two weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec. prior to experimentation.
• 400ml of Murashige and Skoog medium with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 0.01 ⁇ M BA were prepared with 2,4-D concentrations of 10 and 40 ⁇ M. All media were pH adjusted to 5.8, autoclaved at 121°C for 20 minutes, cooled, and each 200 ml portion was poured into 8, 100mm x 15 mm petri dishes.
• a two treatment, random block experimental design with four replications, with one petri dish per replication and 5 fronds per petri dish was used.
• 5 individual duckweed fronds were placed abaxial side down on each plate of medium.
• the plates were incubated at 23°C, for 27 days under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec.
• the duckweed tissue was transferred to fresh media of the same type and incubation was continued another 4 weeks under the same temperature and light culturing conditions.
• Type II Type II
• Type III Type III callus. All three callus types were transferred to fresh medium of identical C coonmposition from that they had been on, and incubation on identical culturing conditions was continued with four week subcultures. After two more months of culture, Type I and Type III callus on 10 ⁇ M 2,4-D and 0.01 ⁇ M BA established healthy, proliferating callus cultures. Type II callus did not proliferate. Although callus proliferation could be maintained on a four-week subculture schedule, callus decline was noted during the third and fourth weeks of the subculture period.
• Example 5 The subculture schedule to maintain callus proliferation was tested with
• Duckweed fronds were grown in liquid Schenk and Hildebrandt medium containing 1% sucrose for two weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec prior to experimentation.
• 500ml of Murashige and Skoog medium with 3% sucrose, 0.15% Gelrite, and 0.4% Difco Bacto-agar, 30 ⁇ M 2,4-D and 0.02 ⁇ M BA was prepared, the pH adjusted to 5.8, autoclaved at 121°C for 30 minutes, cooled, and poured into 20, 100mm x 15 mm petri dishes.
• a two treatment, random block experimental design with two replications, with five petri dish per replication and 5 fronds per petri dish was used.
• 5 individual duckweed fronds were placed abaxial side down on each plate of medium.
• the plates were incubated at 23°C, for 2 weeks under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec.
• the duckweed tissue on half the plates (10 plates) was transferred to fresh medium of the same composition and incubation was continued under the same conditions as those of the non-transferred tissue. After 4 weeks the tissue was assessed for callus proliferation.
• Three types of callus proliferated Type I, Type II, and Type III.
• Type I and Type III callus were subcultured away from the original fronds and continued in culture on Murashige and Skoog medium with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 10 ⁇ M 2,4-D and 0.01 ⁇ M BA. Proliferating callus was continually subcultured to fresh medium of the same composition at 2 week intervals. Longer intervals between transfer resulted in an abrupt decline in callus health between 2 and 3 weeks. Callus proliferation continued without loss of vigor when a two-week subculture schedule was maintained.
• Duckweed fronds were grown in liquid Schenk and Hildebrandt medium containing 1% sucrose for two weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec prior to experimentation.
• 500ml, each, of Murashige and Skoog and Nitsch and Nitsch media with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 30 ⁇ M 2,4-D, and 0.01 ⁇ M BA were prepared, the pH adjusted to 5.8, autoclaved at 121°C for 30 minutes, cooled, and each used to pour 20, 100mm x 15 mm petri dishes.
• a two treatment, random block experimental design with two replications, with five petri dishes per replication and 5 fronds per petri dish was used.
• 5 individual duckweed fronds were placed abaxial side down on each plate of medium.
• the plates were incubated at 23°C, for 2 weeks under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec. After 2 weeks, the duckweed tissue was transferred to fresh medium of the same composition and incubation was continued under the same conditions.
• Type I and Type III callus were subcultured away from the original fronds and continued in culture on Murashige and Skoog medium with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 10 ⁇ M 2,4-D and 0.01 ⁇ M BA. Proliferating callus was continually subcultured to fresh medium of the same composition at two-week intervals. Longer intervals between transfer resulted in an abrupt decline in callus health between 2 and 3 weeks. Callus proliferation continued without loss of vigor.
• Example 7 Three different basal media, Murashige and Skoog, Schenk and Hildebrandt, and Gamborg's B5 (Gamborg et al., In Vitro 12, 473 (1976)) were tested to compare their relative efficacy for callus induction and growth of Lemna gibba G3.
• Duckweed fronds were grown in liquid Schenk and Hildebrandt medium containing 1% sucrose for two weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec prior to experimentation.
• 500ml, each, of the three media were prepared with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 30 ⁇ M 2,4-D and 0.02 ⁇ M BA, the pH adjusted to 5.8, and autoclaved at 121°C for 30 minutes, cooled, and each portion was used to pour 20, 100mm x 15mm petri dishes.
• a three treatment, random block experimental design with two replications, with five petri dishes per replication and 5 fronds per petri dish was used.
• 5 individual duckweed fronds were placed abaxial side down on each plate of medium.
• the plates were incubated at 23°C, for 2 weeks under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 ⁇ sec. After 2 weeks, the duckweed tissue was transferred, to fresh medium of the same composition and incubation was continued under the same conditions.
• Type I and Type III callus were subcultured away from the original fronds and continued in culture on Murashige and Skoog medium with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 10 ⁇ M 2,4-D and 0.01 ⁇ M BA. Proliferating callus was continually subcultured to fresh medium of the same composition at two-week intervals. Longer intervals between transfer resulted in an abrupt decline in callus health between 2 and 3 weeks. Callus proliferation continued without loss of vigor.
• Example 8 Four basal media: Murashige and Skoog (MS), Schenk and Hildebrandt (SH),
• Duckweed fronds were grown in liquid Schenk and Hildebrandt medium containing 1% sucrose for two weeks at 23°C under a 16 hr light 8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m " • sec prior to experimentation.
• 500ml of Murashige and Skoog medium was prepared with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 30 ⁇ M 2,4-D and 0.02 ⁇ M BA, the pH adjusted to 5.8, and autoclaved at 121°C for 30 minutes, cooled, and poured into 20, 100mm x 15mm petri dishes.
• 5 individual duckweed fronds were placed abaxial side down on each plate of medium.
• the 20 plates were incubated at 23°C, for 2 weeks under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec. After 2 weeks, the duckweed tissue was transferred to fresh medium of the same composition and incubation was continued under the same conditions. After 4 weeks Type II callus tissue was used to inoculate liquid medium for callus suspension cultures. For suspension callus establishment, 100 ml, each, of the four basal media, MS, SH, NN, and B5, were prepared with 3% sucrose, 10 ⁇ M 2,4- D, and 0.01 ⁇ M BA.
• the media were adjusted to pH 5.8, four 25 ml aliquots were placed in 125 ml flasks, and all 16 flasks of media were autoclaved at 121°C for 18 minutes. After cooling, each flask was inoculated with 1-2 small pieces of Type II, friable white callus. The flasks were wrapped with aluminum foil and incubated 23°C, for 2 weeks, with constant shaking at 100 rpm, in the dark.
• Duckweed fronds were grown in liquid Schenk and Hildebrandt medium containing 1% sucrose for two weeks at 23°C under a 16 hr light 8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec prior to experimentation.
• six basal media were used: Murashige and Skoog, Schenk and Hildebrand (Schenk and Hildebrandt, Can. J. Bot. 50, 199 (1972)), Nitsch and Nitsch, N6 (Chu et al., Scientia Sinica 18, 659 (1975)), Gamborg's B5, and Hoagland's.
• gibba G3 were used: 30 ⁇ M 2,4-D and 0.02 ⁇ M BA, and 5 ⁇ M 2,4-D and 2 ⁇ M BA.
• 200 ml of each basal medium was prepared with 3% sucrose, 0.15% Gelrite, and 0.4% Difco Bacto-agar.
• the 200 ml was divided into 2, 100 ml portions, each to be used to prepare the two plant growth regulator concentrations.
• the pH of all media was adjusted to 5.8, the media were autoclaved for 30 minutes at 121°C, cooled and 4, 100mm x 15mm petri dishes were poured from each 100 ml portion.
• a 6 media x 2 plant growth regulator combinations, 12 treatment, random block experimental design was used for each duckweed strain tested.
• the design was replicated four times, with one petri dishes per replication and 6 fronds per petri dish.
• 6 individual duckweed fronds were placed abaxial side down on each plate of medium for the larger fronds of Lemna, Spirodela and Wolftella species.
• the small fronds technically prohibited plating of individual fronds, rather, small clumps of fronds were used as the experimental unit.
• the plates were incubated at 23°C, for 4-5 weeks under a 16 hr light 8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec. At this time the fronds were evaluated for general health (judged by color: green to yellow, and vigor of proliferation) and the frequency of callus initiation of the three types: Type I, Type II, and Type III.
• Lemna and Wolffia were the most responsive. All five Lemna gibba strains showed callus induction to varying degrees on MS, B5, and N6 medium containing 5 ⁇ M 2,4- D and 2 ⁇ M BA. Both Lemna minor strains followed the same pattern, with a greater degree of callus induction relative to the Lemna gibba strains. Both Lemna miniscula strains showed a high frequency of callus induction, with proliferation of a white callus somewhat dissimilar to Lemna minor or Lemna gibba.
• Lemna aequinoctialis showed frond curling and swelling at the highest auxin concentrations, but the proliferation of a true callus culture was not observed, indicating that the auxin concentrations used were not high enough.
• Lemna valdiviana did not show callus induction.
• Wolffia arrhiza showed a small amount of callus proliferation on B5 medium with 5 ⁇ M 2,4-D and 2 ⁇ M BA.
• Wolffia brasiliensis and Wolffia columbinana showed callus induction on Hoaglands medium supplemented with 5 ⁇ M 2,4-D and 2 ⁇ M BA.
• Duckweed fronds were grown in liquid Schenk and Hildebrandt medium containing 1% sucrose for two weeks at 23°C under a 16hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/ ⁇ r • sec prior to experimentation.
• three basal media were tested: Murashige and Skoog, Schenk and Hildebrandt, and N6.
• Benzyladenine was used as the cytokinin at a concentration of 1 ⁇ M.
• the auxin concentrations varied with auxin type. For the relatively strong auxins, 2,4-D and dicamba, concentrations were 0, 1, 5, 10 and 20 ⁇ M. For weak auxins, NAA and IBA, the concentrations were 0, 5, 10, 20 and 50 ⁇ M.
• Type I callus induction 2,4-D, dicamba, and NAA all showed some degree of callus induction on MS medium, on N6 medium only 2,4-D and dicamba produced callus. The greatest callus induction was seen on MS medium containing 10 ⁇ M NAA.
• Example 11 Four cytokinins: benzyladenine (BA), kinetin, thidiazuron (TDZ), and 2-iP were tested for their ability to induce callus formation from L. gibba G3 fronds on three different basal media: SH, MS and N6.
• BA benzyladenine
• TDZ thidiazuron
• 2-iP 2-iP
• Duckweed fronds were grown in liquid Schenk and Hildebrandt medium containing 1% sucrose for two weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec prior to experimentation.
• three basal media were tested: Murashige and Skoog, Schenk and Hildebrandt, and N6.
• 2,4-D was used as the auxin at a concentration of 20 ⁇ M.
• the cytokinin concentrations used were 0, 0.05, 0.1, 0.5, 1 and 5 ⁇ M.
• 2400 ml of basal medium were prepared with 2,4-D and the pH adjusted to 5.8.
• the volume was aliquoted as 24, 100 ml portions. To each of these portions, the appropriate amount of cytokinin was added and the medium was adjusted to 0.15% Gelrite, and 0.4% Difco Bacto- agar. The media were autoclaved for 30 minutes at 121°C, cooled and 4, 100mm x 15mm petri dishes were poured from each 100 ml portion. A 3 media x 4 cytokinin types x 6 cytokinin concentrations combinations, 72 treatment, randomized dose-response experimental design was used. The design was replicated two times, with one petri dish per replication and 5 fronds per petri dish. For callus induction, 5 individual duckweed fronds were placed abaxial side down on each plate of medium.
• Example 12 As Lemna minor strains 8744 and 8627 showed greater callus induction and more rapid callus proliferation than L. gibba strains (see Example 9 and Table I), further optimization of culturing conditions was done for L. minor. Variables tested for callus induction included: a) screening of basal medium composition, b) auxin type and concentration screening, and c) cytokinin type and concentration screening. In the basal medium screen, three media were tested: Schenck and
• IBA and dicamba were tested, each at four concentrations: 2, 5, 10 and 20 ⁇ M, for their ability to induce callus formation from L. minor strains 8744 and 8627.
• the basal medium used was MS and media and experimental protocol was basically that followed in Example 10.
• Fronds used in this experiment were grown for 2 weeks prior to plating on callus induction medium under 3 different culture conditions: 1) SH medium without plant growth regulators, 2) F medium with 24 ⁇ M 2,4-D and 2 ⁇ M 2-iP, and 3) SH medium with 24 ⁇ M 2,4-D and 2 ⁇ M 2-iP. Fronds were separated, the roots cut off and then plated on induction medium. The fronds were incubated under conditions given in Example 8 for 6 weeks at which time cultures were evaluated for the presence or absence of callus induction, the degree to which the callus proliferated, and the basic morphology of the callus present.
• cytokinin type and concentration experiment four cytokinins: BA, kinetin, 2-iP, and thidiazuron were tested, each at five concentrations: 0.05, 0.1, 0.5, 1 and 5 ⁇ M, for their ability to induce callus formation from L. minor strains 8744 and 8627.
• the basal medium used was MS and the media and experimental protocol were basically as described in Example 11. Fronds used in this experiment were grown for 2 weeks prior to plating on callus induction medium under 3 different culture conditions: 1) SH medium without plant growth regulators, 2) F medium with 24 ⁇ M 2,4-D and 2 ⁇ M 2-iP, and 3) SH medium with 24 ⁇ M 2,4-D and 2 ⁇ M 2-iP.
• Fronds were separated, the roots cut off and then plated on induction medium. The fronds were incubated under conditions given in Example 8 for 6 weeks at which time cultures were evaluated for the presence or absence of callus induction, the degree to which the callus proliferated, and the basic morphology of the callus.
• Basal medium composition was tested for its effect on callus proliferation and long term establishment using L. minor strains 8627 and 8644.
• MS Three basal medium compositions were tested for their ability to maintain healthy callus growth: MS, F-medium and half-strength SH. All media contained 3% sucrose and were gelled with 0.4% Difco Bacto-agar and 0.15% Gelrite.
• the MS medium was supplemented with 1 ⁇ M 2,4-D, 2 ⁇ M BA; the half-strength SH medium was supplemented with 1 ⁇ M BA; and the F-medium was supplemented with 9 ⁇ M 2,4-D and 1 ⁇ M 2-iP.
• Callus cultures from both strain 8744 and strain 8627 proliferated in a previous callus induction medium as in Example 12 were used for this experiment. Callus was grown for a two-week subculture period and scored for growth, color and general health.
• L. minor 7501, 8626, and 8745 The callus induction system developed in the previous Examples was followed: Murashige and Skoog basal medium supplemented with 3% sucrose. 5 ⁇ M 2,4-D and 2 ⁇ M BA, and gelled with 0.4% Difco Bacto-agar and 0.15% Gelrite was used for callus induction.
• Fronds were grown on liquid SH medium devoid of plant growth regulators and supplemented with 1% sucrose prior to plating on callus induction medium. Fronds were plated onto callus induction medium and scored 5 weeks later for relative frequencies of callus induction and relative rates of callus proliferation.
• strains 8626 and 8745 callus induction did not occur during the 5-week induction period, however subsequent culture did yield a low frequency of callus proliferation.
• the morphology and color of callus from strains 8626 and 8745 was quite similar to that proliferated from 8744 and 8627 and proliferated quite well when transferred to callus maintenance medium.
• Strain 7501 showed a low frequency of callus induction, with callus similar in morphology to that produced from strains 8626 and 8745.
• Callus induction, growth and frond regeneration from duckweed plants is accomplished through incubation on the appropriate medium and manipulation of the plant growth regulator types and concentrations at specific developmental stages to promote callus formation, growth and reorganization to fully differentiated plants.
• the preferred media for callus induction are N6 and MS, most preferred is MS.
• Fronds are incubated in the presence of both an auxin and a cytokinin, the preferred auxins are NAA and 2,4-D and the preferred cytokinins are BA and TDZ.
• concentrations of these plant growth regulators vary over a broad range.
• the preferred concentrations are 5- 20 ⁇ M, the most preferred are 5-10 ⁇ M, and for the cytokinins, the preferred concentrations are 0.5-5 ⁇ M, the most preferred are 0.5-1 ⁇ M.
• the fronds are incubated for an induction period of 3-5 weeks on medium containing both plant growth regulators with callus proliferating during this time.
• the preferred media are as for callus induction, but the auxin concentration is reduced.
• the preferred concentrations are 1 -5 ⁇ M, and for cytokinins the preferred concentrations are 0.5-1 ⁇ M.
• the subculture period is also reduced from 4-5 weeks, for callus induction, to 2 weeks for long-term callus growth.
• Callus growth can be maintained on either solid medium gelled with agar, Gelrite, or a combination of the two, with the preferred combination of 0.4% Difco Bacto-agar and 0.15% Gelrite, or on liquid medium. Callus cultures can be maintained in a healthy state for indefinite periods of time using this method.
• pre-callus induction morphology was readily apparent in several strains, including Wolffia arrhiza 8853, 9000, 9006 and Wolffia brasilensis 7581. With these strains, frond thickening was apparent, a response frequently seen in fronds before callus for tmuation becomes apparent and indicates that the auxin concentrations used was insufficient to support callus proliferation.
• Transformation This section covers experiments pertaining to the methods used for actual gene transfer. There are three sections: (1) Transformation of fronds using the gene gun, (2) Agrobacterium-mediated transformation using duckweed fronds, and (3) Agrobacterium-mediated transformation using duckweed callus.
• the transformation of fronds experiments were used to optimize the parameters affecting actual gene transfer: (a) bacterial growth, (b) inclusion of acetosyringone, (c) bacterial concentration, (d) solution for resuspending bacteria and the effect of osmotic shock, (e) co-cultivation medium for fronds and callus, (f) duration of the time of inoculation, (g) co-cultivation time for fronds and callus, and (h) light conditions during co-cultivation.
• the protocol developed with fronds was applied to transform the callus cultures obtained using the optimized tissue culture procedure. It is this transformed callus that is taken on to selection and then through regeneration to obtain transformed fronds.
• frond proliferation 60 ml of high salt medium (De Fossard, TISSUE CULTURE FOR PLANT PROPAGATORS 132-52 (1976)) supplemented with 3% sucrose and 0.8% agar was prepared, the pH adjusted to 5.8, autoclaved for 20 minutes at 121°C, cooled, and used to pour 6, 60mm x 15mm petri dishes. One frond was inoculated to each petri dish. The fronds were grown for two weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40
• plasmid pRT99 Topfer et al., Nucleic Acid Res. 16, 8725 (1988)
• the plasmid, pRT99 encodes the neomycin phosphotransferase gene and the ⁇ - glucuronidase gene (GUS; Jefferson et al., EMBO J. 6. 3901 (1987)), both under the control of CaMV35 S promoters.
• GUS Jefferson et al., EMBO J. 6. 3901 (1987)
• Duckweed fronds were turned abaxial side up and bombarded with the DNA coated microcarriers at four pressure levels of helium: 800, 600, and 400 lbs/sq. inch. Histochemical staining for GUS activity using 5-bromo-4-chloro-3-indolyl- ⁇ -D- glucuronic acid (X-gluc) as the substrate following the method of Stomp (Histochemical localization of beta-glucoronidase, in GUS PROTOCOLS 103-1 14 (S.R. Gallagher ed. 1991)) was done 24 hours after bombardment.
• the frequency of GUS positive staining centers was directly proportional to the pressure used for bombardment, with the greatest number of GUS expressing cells found in the 800 psi treatment, with frequency ranging from 4-20 staining cells/frond. In all treatments, bombardment resulted in the destruction of more than half the fronds.
• Example 19 Fronds of Lemna gibba G3 were subjected to microprojectile bombardment to test the effect of microcarrier size on the frequency of foreign gene expression.
• frond proliferation 200 ml of high salt medium supplemented with 3% sucrose and 0.8% agar was prepared, the pH adjusted to 5.8, autoclaved for 20 minutes at 121°C, cooled, and used to pour 20, 60mm x 15mm petri dishes. One frond was inoculated to each petri dish. All fronds were grown for two weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec. Two microcarrier size 1.0 and 1.6 ⁇ m were tested at 3 helium pressure levels: 400, 800, and 1200 psi, using a PDS-1000/He gene gun manufactured by DuPont. Gold microcarriers were prepared and pRT99 DNA was precipitated onto the microcarriers following methods supplied by the manufacturer (Bio-Rad).
• Transgenic duckweed plants are regenerated from duckweed callus transformed by ballistic bombardment.
• Type I callus cultures are grown as described in Example 42 below.
• 20-30 duckweed callus pieces, approximately 2-4 mm in diameter, are spread evenly across the bombardment area on MS medium (MS medium described in Example 42).
• Gold particles 1.6 ⁇ M in diameter
• bombardment helium pressure of 800 psi
• the DNA for bombardment consists of an expression plasmid containing the gene of interest (e.g., GUS, another marker gene, a gene encoding a mammalian protein, or a gene encoding a bacterial, fungal, plant or mammalian enzyme) and a gene encoding a selectable marker gene, e.g., nptll (kanamycin resistance), hptll (hygromycin resistance), sh ble (zoecin resistance), and bar (phosphinotricin resistance), as well as other sequences necessary for gene expression (e.g., promoter sequences, termination sequences).
• GUS another marker gene, a gene encoding a mammalian protein, or a gene encoding a bacterial, fungal, plant or mammalian enzyme
• a selectable marker gene e.g., nptll (kanamycin resistance), hptll (hygromycin resistance), sh ble (zoecin resistance), and bar
• the callus is incubated in the dark for two days (or longer if necessary), followed by incubation under a light intensity of 3-5 ⁇ mol/m" ⁇ sec for 4-6 weeks. Callus is transferred to fresh medium every two weeks, with the selectable agent added to the medium 2-4 weeks post-bombardment. Selection of resistant callus is continued for 8-16 weeks, until fully resistant callus is produced. Regeneration of transgenic fronds and plants is carried out as described in Example 42.
• Example 21 Duckweed fronds of Lemna gibba G3 were used to test the susceptibility of duckweed to Agrobacterium tumefaciens using two different media for co-cultivation, Schenk and Hildebrandt and Murashige and Skoog.
• Agrobacterium tumefaciens strain AT656 and non-virulent A. tumefaciens strain A136 were used to inoculate the duckweed fronds.
• Strain AT656 is constructed from strain EHA105 (Hood et al., Transgenic Res. 2, 208 (1993)) which contains the pTiBo542 vir region on a disarmed pTiBo542 plasmid.
• the T-DNA is carried on a binary plasmid, pCNL56 (Li et al., PI. Mol. Biol. 20, 1037 (1992)).
• This binary plasmid is derived from pBIN19, and as modified carries a neomycin phosphotransferase gene under the control of the nopaline synthetase promoter and a nopaline synthetase terminator, and a ⁇ -glucuronidase (GUS) gene (Janssen and Gardner, Plant Mol. Biol. 14, 61 (1989)) under the control of the mas2'-CaMV35S promoter and an octopine synthetase terminator.
• the GUS coding region contains an intron within the coding sequence of the gene to prevent bacterial expression of GUS (Vancanneyt et al., Mol. Gen. Genet. 220, 245 (1990)).
• Strain A136 is derived from the broad host range strain, C58. When C58 is grown at temperatures above 30°C it loses its Ti-plasmid becoming avirulent A136. These two strains, AT656 and A136, were grown overnight on AB minimal medium (Chilton et al., Proc. Nat. Acad. Sci. USA 71, 3672 (1974)) solidified with 1.6% agar and supplemented with 100 ⁇ M acetosyringone at 28°C.
• Duckweed fronds were grown in liquid Hoagland's medium containing 3% sucrose for two weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec prior to experimentation.
• 500 ml of Schenk and Hildebrandt medium containing 1% sucrose and 0.6% agar was prepared, the pH adjusted to 5.6, autoclaved at 121°C for 30 minutes, and cooled.
• Five-hundred ml of Murashige and Skoog medium containing 3% sucrose and 0.6% agar were also prepared, the pH adjusted to 5.8, autoclaved at 121°C for 30 minutes, and cooled.
• a filter-sterilized solution of acetosyringone was added to a final, medium concentration of 20 mg/L.
• the bacteria from one, 100 mm x 15mm petri dish were resuspended for at least one hour prior to use in 100 ml of the following solution (Hiei et al., The Plant J.
• duckweed fronds were floated in the bacterial solution for several minutes.
• co-cultivation the fronds were transferred to either Schenk and Hildebrandt or Murashige and Skoog medium as described above. The fronds were incubated at 23 C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m ⁇ sec for four days. The fronds were then transferred to fresh medium of the same composition except that acetosyringone was absent and 500 mg/L of timentin and 50 mg/L kanamycin sulfate were added to the medium.
• Agrobacterium tumefaciens strain AT656 was used and was grown overnight at 28°C on AB minimal medium (Chilton et al., Proc. Nat. Acad. Sci
• the clumps were separated into individual fronds, the fronds were turned abaxial side up, and fronds were wounded one of two ways: 1) cut transversely across the frond centrum, thus cutting through the adjacent meristematic regions from left to right, or 2) cut on each side of the centrum, thus cutting longitudinally through each meristematic region.
• both classes of wounded fronds were floated on: 1) resuspended AT656 or 2) in the resuspension fluid without the bacteria.
• fronds were left floating for 10-30 minutes.
• fronds were transferred to Murashige and Skoog medium as described above with 3% sucrose, 20 ⁇ M 2,4-D, 2 ⁇ M BA, 100 ⁇ M acetosyringone, and 0.6% agar.
• the fronds were incubated at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec for four days.
• a frond subsample was stained for GUS following the procedure of Stomp (Histochemical localization of beta-glucoronidase, in GUS PROTOCOLS 103-1 14 (S.R. Gallagher ed. 1991).
• Fronds of Lemna gibba G3 were used to determine the effect of inoculation time for wounded fronds in bacterial resuspension medium on the frequency of GUS expression after co-cultivation.
• Duckweed fronds were grown in liquid Hoagland's medium containing 1% sucrose to a density of approximately 120 fronds per 25 ml of medium in a 125 ml flask at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec prior to experimentation.
• 1500 ml of Schenk and Hildebrandt medium with 1% sucrose and 0.6% agar was prepared, the ⁇ % pH adjusted to 5.6, autoclaved at 121°C for 30 minutes, and cooled.
• a filter-sterilized solution of acetosyringone was added to a final, medium concentration of 20 mg/L.
• Sixty, 100mm x 15mm petri dishes were poured from the cooled medium.
• a randomized block experimental design with 4 inoculation time treatments, with 3 replications, with 5 petri dish per replication, and 25 fronds per petri dish was used.
• Agrobacterium tumefaciens strain AT656 was used and was grown overnight at 28°C on AB minimal medium containing 50 mg/L kanamycin sulfate and 20 mg/L acetosyringone.
• the bacteria from one 100mm x 15mm petri dish were resuspended as described in Example 21.
• fronds were separated from clumps, each turned abaxial side up and wounded with a sterile scalpel in the meristematic regions, then transferred to bacterial suspensions and incubated for 15, 30, 45, or 60 minutes.
• fronds were transferred to Schenk and Hildebrandt co-cultivation medium as described above. All 60 petri dishes were incubated at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec for six days. The three replicates were done over a four-day period.
• C58sZ707pBI121 is a disarmed, broad host range C58 strain (Hepburn et al, J. Gen. Microbiol. 131, 2961 (1985)) into which pBI121 has been transferred.
• the binary plasmid, pBI121 is derived from pBIN19 and its T-DNA encodes a neomycin phosphotransferase gene under the control of the nopaline synthetase promoter and a nopaline synthetase terminator, and a ⁇ -glucuronidase (GUS) gene under the control of a CaMV35S promoter and an octopine synthetase terminator.
• GUS ⁇ -glucuronidase
• AT656 was streaked on AB minimal medium containing kanamycin sulfate at 50 mg/L and C58sZ707pBI121 was streaked on AB minimal medium containing streptomycin at 500 mg/L, spectinomycin at 50 mg/L and kanamycin sulfate at 50 mg/L. Both bacterial strains were grown overnight at 28°C.
• Duckweed fronds were grown in liquid Hoagland's medium containing 1% sucrose for four weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec prior to experimentation.
• 500ml of Schenk and Hildebrandt medium with 1% sucrose and 0.6% agar was prepared, the pH adjusted to 5.6, autoclaved at 121°C for 30 minutes, and cooled.
• a filter-sterilized solution of acetosyringone was added to a final, medium concentration of 20 mg/L. Twenty, 100 mm x 15 mm petri dishes were poured from the cooled medium.
• a randomized block, experimental design with 2 bacterial strain treatments, with 2 replications, with 5 petri dish per replication, and 25 fronds per petri dish was used.
• bacteria from one A sBr plate of each strain were resuspended as described in Example 21.
• fronds were separated from clumps, each turned abaxial side up and wounded with a sterile scalpel in the meristematic regions, then transferred to bacterial suspensions and incubated for 15-30 minutes.
• fronds were transferred to Schenk and Hildebrandt co-cultivation medium as described above. All 20 petri dishes were incubated at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec for six days. A subsample of fronds was taken at 6 days of co-cultivation and stained for GUS expression.
• Fronds of Lemna gibba G3 were used to determine the effect of Agrobacterium strain, foreign gene construct, and frond pre-treatment on the frequency of GUS expression after co-cultivation.
• Two Agrobacterium tumefaciens strains were used: AT656 and EHAlOlpJRl .
• EHAlOlpJRl is a binary Agrobacterium tumefaciens strain containing a disarmed pTiBo542 plasmid harboring the hypervirulence region of wild-type strain, Bo542, and a small binary plasmid harboring a hygromycin phosphotransferase gene under the control of an alcohol dehydrogenase 1 enhanced, CaMV35S promoter and a ⁇ - glucuronidase gene constructed as in AT656. These two strains were streaked on potato dextrose agar with 50 mg/L kanamycin and grown overnight at 28°C.
• Duckweed fronds were grown on liquid Schenk and Hildebrandt medium containing 1% sucrose with and without 10 ⁇ M indoleacetic acid (IAA), a concentration sufficient to increase proliferation rate. Fronds were grown in 25 ml aliquots of medium in 125 ml flasks, at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 ⁇ sec.
• IAA indoleacetic acid
• a randomized block, experimental design with 2 bacterial strain treatments x 2 frond growth media, with 5 replications, with one petri dish per replication, and 20 fronds per petri dish was used.
• bacteria of each strain were separately resuspended as described in Example 21.
• individual fronds were separated from clumps, each turned abaxial side up and wounded with a sterile scalpel in the meristematic regions, then transferred to bacterial suspensions of either AT656 or EHAlOlpJRl, and incubated for 10-15 minutes.
• the fronds were co-cultivated for 4 days at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m " • sec.
• the fronds from two plates from each of the four treatments were stained for GUS expression.
• Table III presents the results of GUS staining.
• Fronds of Lemna gibba G3 were co-cultivated for five different times: 12.5, 18.5, 40.5, 82, and 112 hours, with bacterial strain AT656 to test the effect of co- cultivation time on GUS expression after co-cultivation.
• Duckweed fronds were grown for two weeks on liquid Schenk and Hildebrandt medium containing 1%> sucrose and 10 ⁇ M indoleacetic acid at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec prior to experimentation.
• 750 ml of Schenk and Hildebrandt medium with 1% sucrose, 0.8% agar, 10 ⁇ M indoleacetic acid, and 20 mg/L acetosyringone was prepared, the pH was adjusted to 5.6, the medium autoclaved at 121°C for 30 minutes, and cooled.
• a filter-sterilized solution of acetosyringone and indoleacetic acid was added to the final medium concentration. Thirty, 100 mm x 15 mm petri dishes were poured from the cooled medium. Bacterial strain AT656 was streaked on potato dextrose agar with 50 mg/L kanamycin sulfate and grown overnight at 28°C.
• Fronds were then transferred to the bacterial resuspension solution and incubated for approximately 10-15 minutes. For co-cultivation, fronds were transferred to solid
• GUS expression Before 40 hours, no GUS expression was detectable. By 3.5 days (82 hours) GUS expression was readily detectable. Longer co-cultivation did not significantly increase the frequency, intensity, or tissue association pattern of GUS expression in duckweed fronds. It was concluded that 3.5-4 days is the shortest co- cultivation time that will give the maximum frequency of gene transfer in duckweed fronds.
• Example 27 Bacteria of strain AT656, grown on three different bacterial media: AB minimal, potato dextrose, and mannitol glutamine Luria broth, were used to co- cultivate Lemna gibba G3 fronds, that had been grown with and without indoleacetic acid prior to co-cultivation, in light and in the dark to test the effects of these treatments on GUS expression following co-cultivation.
• Lemna gibba G3 fronds were grown for two weeks on liquid Schenk and
• a filter-sterilized solution of kanamycin sulfate and acetosyringone was added to the cooled media to final medium concentrations of 50 mg/L and 20 mg/L, respectively. AT656 was streaked on these three media and incubated overnight at 28°C.
• Bacterial medium has a significant effect on the frequency of GUS expression after 4 days of co-cultivation.
• AB medium gave the lowest frequency of GUS expression and PDA the highest.
• Growing fronds on indoleacetic acid prior to inoculation increased the frequency of k GLUS eexxpression after co-cultivation.
• the presence of light during co-cultivation did not significantly affect the frequency of GUS expression after co-cultivation in treatments using fronds grown without indoleacetic acid, however, co-cultivation in the dark did increase the frequency of GUS expression in treatments that used fronds grown in the presence of indoleacetic acid.
• Averaging frequencies from PDA and MGL across the duckweed fronds grown on Schenk and Hildebrandt medium with indoleacetic acid gives a frequency of GUS expression in meristematic tissue of approximately 17%.
• Lemna gibba G3 fronds were grown for 17 days on Schenk and Hildebrandt medium containing 1% sucrose and 10 ⁇ M indoleacetic acid at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec.
• 150 ml of Schenk and Hildebrandt medium containing 1% sucrose, 1 % agar, 10 ⁇ M indoleacetic acid, and 20 mg/L acetosyringone was prepared, the pH adjusted to 5.6, autoclaved for 30 minutes, and cooled.
• Fronds of Lemna gibba G3 were used to determine the effect of bacterial resuspension solutions, the osmotic potential of these solutions, and frond wounding on the frequency of GUS expression following co-cultivation.
• the cooled medium was used to pour 72, 100 mm x 15 mm petri 0 dishes.
• Agrobacterium strain AT656 was streaked onto AB minimal medium containing 20 mg/L acetosyringone and 50 mg/L kanamycin sulfate and grown overnight at 28°C.
• Fronds of Lemna gibba G3 were used to test the effect of bacterial concentrations during inoculation on the frequency of GUS expression following co- cultivation.
• Agrobacterium strain AT656 was streaked on half-strength potato dextrose agar - mannitol glutamine Luria broth medium with 1.6% Difco Bacto-agar, 20 mg/L acetosyringone, and 50 mg/L kanamycin sulfate and grown overnight at 28°C.
• a randomized block experimental design with 10 bacterial concentration treatments, with 3 replications, with one petri dish per replication and 20 individual fronds or frond clumps per petri dish was used.
• bacteria from one petri dish were resuspended as described in Example 21.
• This bacterial solution constituted the "undiluted" sample and was the beginning of a serial dilution series for the following dilutions: 1/3, 10 "1 , 1/33, 10 "2 , 1/333, 10 "3 , 1/3333, 10 "4 , 10 "5 .
• the 1/3 dilution had an OD540nm of 1.006, which corresponded to approximately 1.6 x 10 9 bacteria/ml.
• a randomized block experimental design with 4 media treatments with four replications, with one petri dish per replication and 20 fronds per petri dish was used.
• the bacteria from one petri dish were resuspended for one hour prior to use in 100 ml of filter-sterilized SH medium with 0.6M mannitol and 20 mg/L acetosyringone at pH 5.6.
• individual fronds were separated from clumps and floated in the resuspended bacteria for 8-10 minutes.
• the fronds were transferred to co-cultivation medium described above (MSI, MS2, MS3, SH). Fronds were co-cultivated at 23°C in the dark for four days. After four days of co-cultivation, all fronds were stained for GUS expression.
• the frequency of fronds showing GUS expression ranged from 80-90% across all treatments. Co-cultivation medium did not have a significant effect on this frequency.
• the intensity of GUS staining ranged from light to intense. Staining was associated with root tips, stems, broken ends of stems and wounds, meristematic regions, and the frond margins.
• Example 32 Frond transformation using Agrobacterium is accomplished through manipulation of the cell division rate of the fronds prior to inoculation, the medium on which the Agrobacteria are grown, optimization of co-cultivation parameters including secondary metabolites such as acetosyringone, the concentration of the Agrobacteria, the osmolarity of the inoculation fluid, the duration of the co- cultivation period, and the light intensity of the co-cultivation period.
• secondary metabolites such as acetosyringone
• fronds are grown on medium containing an auxin that increases the proliferation rate of the fronds, with NAA, IBA and IAA being the preferred auxins and the preferred concentrations ranging from 0.2-1 ⁇ M.
• Agrobacteria are grown on a medium without rich nutrient supplements and including such secondary metabolites as acetosyringone, with potato dextrose agar and mannitol glutamine Luria broth as preferred media.
• the frequency of transformation is determined by the composition of the inoculating fluid, with the preferred fluid being MS or SH basal salts supplemented with 0.6 M mannitol and 100 ⁇ M acetosyringone.
• the concentration of Agrobacteria resuspended in this inoculating fluid also affects the frequency of transformation, with the preferred concentration on the order of 1 x 10 9 bacteria per ml.
• Inoculation time can vary with the preferred time ranging from 2-20 minutes.
• Co-cultivation time also affects the frequency of transformation, with a time of 3-4 days being preferred. Co-cultivation can be carried on under light or dark conditions, with darkness (e.g., subdued light) being preferred.
• MS and SH are the preferred media.
• Decontamination of the fronds from infecting Agrobacteria is done using the approp Lrliate antibiotics at high concentrations, typically 100-500 mg/L, with frequent transfer of infected tissue, the preferred method being transfer to fresh medium with antibiotic every 2-4 days. Incubation under low light intensity, the preferred range being 1-5 ⁇ mol/m 2 . sec, for an initial resting/recovery period of 3-6 weeks is preferred.
• Selection by growth in the presence of the selection agent can be initiated at variable times, with the preferred time being 1-3 weeks after inoculation. Initial selection under reduced light levels and low selection agent concentration is also preferred, with light levels of 1-5 ⁇ mol/nr.sec and low concentration ranges appropriate for the selection agent as determined from toxicity studies for the specific agent. For kanamycin sulfate, the typical range is 2-10 mg/L.
• Agrobacterium tumefaciens strain AT656 was streaked on half-strength potato dextrose agar mixed with half-strength mannitol glutamine Luria broth medium containing 20 mg/L acetosyringone and 50 mg/L kanamycin sulfate overnight at 28°C.
• Example 34 Twenty strains of duckweed from the 4 genera of the Lemnaceae were tested for their ability to give GUS expression following co-cultivation with Agrobacterium strain AT656 using the transformation protocol developed with L. gibba G3. The twenty strains were: Wolffiella lingulata strains 8742 and 9137, Wl. neotropica strains 7279 and 8848, Wl. oblongata strains 8031 and 8751. Wolffia arrhiza strains 7246 and 9006, Wa. australiana 7317, Wa. brasiliensis strains 7397, 7581, and 8919, Wa. columbiana strains 7121 and 7918, Spirodela intermedia 7178, S. polyrrhiza strains 7960 and 8652, S. punctata strains 7488 and 7776, and L. gibba G3.
• the bacterial strain, AT656 was streaked on potato dextrose agar with 20 mg/L acetosyringone and 50 mg/L kanamycin sulfate and grown overnight at 28°C.
• the bacteria from one petri dish were resuspended for at least one hour prior to use in Schenk and Hildebrandt medium with 0.6M mannitol, 20 mg/L acetosyringone, pH of 5.6 that was filter-sterilized before use.
• a randomized block experimental design with 20 duckweed strain treatments, with 3 replications, with one petri dish per replication and 20 individual fronds or frond clumps per petri dish was used.
• Type I callus produced from Lemna gibba G3 fronds was used to test its ability to give GUS expression using the optimized transformation protocol developed with L. gibba G3 fronds and to test the effect of vacuum infiltration.
• Type I callus was produced by growing fronds on solid Murashige and Skoog medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 5 ⁇ M 2,4- dichlorophenoxyacetic acid (2,4-D), and 2 ⁇ M benzyladenine (BA). Callus induction and all subsequent culture was at 23°C and under a 16 hr light/8 hr dark photoperiod 1° with light intensity of approximately 40 ⁇ mol/m " • sec. After 4 weeks of callus induction, Type I callus clumps were separately cultured on the same medium with the 2,4-D concentration reduced to 1 ⁇ M. The callus was subcultured to fresh medium every two weeks until sufficient callus was proliferated for experimentation. For co-cultivation, 400 ml of solid Murashige and Skoog medium (MS) with
• sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, l ⁇ M 2,4-D, and 2 ⁇ M BA was prepared, the pH adjusted to 5.6, autoclaved at 121°C for 20 minutes, and cooled.
• a filter-sterilized solution of acetosyringone was added to a final, medium concentration of 20 mg/L.
• the cooled medium was used to pour 16, 100 mm x 15 mm petri dishes.
• Agrobacterium strain AT656 was streaked on potato dextrose agar with 20 mg/L acetosyringone and 50 mg/L kanamycin sulfate and grown overnight at 28°C.
• a randomized block experimental design with two vacuum infiltration treatments with four replications with two petri dishes per replication and ten callus pieces per petri dish was used.
• the bacteria were resuspended in filter-sterilized Schenk and Hildebrandt medium containing 0.6M mannitol and 20 mg/L of acetosyringone at pH 5.6 for at least one hour before use. Inoculation with bacteria was done with and without vacuum infiltration. Without vacuum infiltration, small pieces of Type I callus were placed in the bacterial solution for 10 minutes, then blotted and transferred to MS co-cultivation medium as described above. With vacuum infiltration, the callus was placed in bacterial solution, a vacuum of 10 inches of mercury applied for 10 minutes, then the callus was blotted and transferred to MS co-cultivation medium. All dishes were co-cultivated in the dark at 23°C.
• Type I callus was produced by growing Lemna gibba G3 fronds on solid
• MS Murashige and Skoog medium
• MSI Murashige and Skoog medium
• MS2 MS medium with 20 ⁇ M 2,4-D and 1 ⁇ M BA
• MS3 MS medium with 1 ⁇ M 2,4-D and 2 ⁇ M BA
• SH Schenk and Hilderbrandt medium
• a randomized block experimental design with 4 co-cultivation media treatments with two replications with one petri dish per replication and 20 callus pieces per petri dish was used.
• the bacteria from one petri dish were resuspended in filter sterilized SH medium containing 0.6M mannitol and 20 mg/L acetosyringone at pH 5.6 for at least one hour prior to use.
• Type I callus pieces were placed in bacterial solution for 8 minutes, blotted and then transferred to the four different co-cultivation media. All plates were co-cultivated at 23°C in the dark for four days. After co-cultivation, all callus was stained for GUS expression following the procedure of Stomp et al.
• Example 37 Two different co-cultivation times, two and four days, were tested for their effect on the frequency of GUS expression following co-cultivation of Type I callus with Agrobacterium strain AT656.
• Type I callus was produced by growing Lemna gibba G3 fronds on solid
• a randomized block experimental design with two co-cultivation time treatments with two replications with four petri dishes per replication and 10 callus pieces per petri dish was used.
• the bacteria were resuspended in filter-sterilized Schenk and Hildebrandt medium containing 0.6M mannitol and 20 mg/L acetosyringone at pH 5.6 for at least one hour before use.
• Type I callus pieces were placed in bacterial solution.
• the pieces were blotted, then transferred to MS co-cultivation medium described above. All plates 7 ⁇ were co-cultivated in the dark at 23°C for either two or four days. After either two or four days of co-cultivation, all callus was stained for GUS expression.
• Example 38 A different gene construct was used to test the efficacy of the Type I callus co- cultivation protocol with another Agrobacterium strain, C58sZ707pBI121.
• Type I callus was produced by growing Lemna gibba G3 fronds on solid Murashige and Skoog medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 5 ⁇ M 2,4-D, and 2 ⁇ M BA. Callus induction and all subsequent culture was at 23°C and under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec. After 4 weeks of callus induction, Type I callus clumps were separately cultured on the same medium with the 2,4-D concentration reduced to 1 ⁇ M. The callus was subcultured to fresh medium every two weeks until sufficient callus was proliferated for experimentation. For co-cultivation, 400 ml of solid Murashige and Skoog medium (MS) with
• Agrobacterium strain C58sZ707pBI121 was streaked on potato dextrose agar with 20 mg/L acetosyringone, 500 mg/L streptomycin sulfate, 50 mg/L spectinomycin, and 50 mg/L kanamycin sulfate and grown overnight at 28°C.
• a randomized block experimental design with one bacterial strain treatment with four replications with four petri dishes per replication and 10 callus pieces per petri dish was used.
• the bacteria from one petri dish were resuspended in filter-sterilized Schenk and Hildebrandt medium containing 0.6M mannitol and 20 mg/L of acetosyringone at pH 5.6 for at least one hour before use.
• Type I callus pieces were placed in bacterial solution for 8-10 minutes.
• co-cultivation the pieces were blotted and then transferred to MS co- cultivation medium described above. All callus was co-cultivated in the dark at 23°C for two days.
• Example 39 Type II callus and Type III callus were tested for their ability to give GUS expression following co-cultivation in the presence of Agrobacterium strain AT656.
• Both callus types were induced by culturing Lemna gibba G3 fronds on solid Murashige and Skoog medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 30 ⁇ M 2,4-D and 0.02 ⁇ M BA at 23°C under a 16 hr light 8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec.
• Type II callus and Type III callus were separated from the original fronds and transferred to solid Murashige and Skoog medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 10 ⁇ M 2,4-D, and 0.01 ⁇ M BA for callus maintenance under the same temperature and light conditions.
• the callus was subcultured to fresh medium every two weeks until sufficient callus was proliferated for experimentation.
• Agrobacterium strain AT656 was streaked on potato dextrose agar containing 20 mg/L acetosyringone and 50 mg/L kanamycin sulfate and grown overnight at 28°C.
• 200 ml of Murashige and Skoog medium (MS) with 3% sucrose, 0.15% Gelrite and 0.4% Difco Bacto-agar, 10 ⁇ M 2,4-D and 0.02 ⁇ M BA was prepared, the pH adjusted to 5.6, autoclaved at 121°C for 20 minutes, and cooled.
• a filter-sterilized solution of acetosyringone was added to the cooled medium to a final concentration of 20 mg/L.
• the cooled medium was used to pour 8, 100 mm x 15 mm petri dishes.
• a randomized block experimental design with two callus type treatments was used. Forty clumps of green callus, transferred evenly to 4 petri dishes, and 9 clumps of white callus, transferred evenly to 4 petri dishes, were inoculated.
• bacteria were resuspended in filter-sterilized Schenk and Hildebrandt medium containing 0.6M mannitol and 20 mg/L acetosyringone at pH 5.6 for at least one hour before inoculation.
• pieces of green callus and white callus were dipped in the bacterial solution for 2-5 minutes.
• callus pieces were blotted then transferred as clumps to MS co-cultivation medium described above. All callus was incubated at 23°C in the dark for two days.
• Type I callus established from two different fast-growing strains of Lemna gibba (strain 6861 and 7784) and one strain of Lemna minor were co-cultivated with AT656 to determine the frequency of transformation with the protocol established using Lemna gibba G3.
• Agrobacterium strain AT656 was streaked on potato dextrose agar containing 20 mg/L acetosyringone and 50 mg/L kanamycin sulfate and grown overnight at 28°C.
• 200 ml of Murashige and Skoog medium (MS) with 3% sucrose, 0.15% Gelrite and 0.4% Difco Bacto-agar, 10 ⁇ M 2,4-D and 0.02 ⁇ M BA was prepared, the pH adjusted to 5.6, autoclaved at 121°C for 20 minutes, and cooled.
• a filter-sterilized solution of acetosyringone was added to the cooled medium to a final concentration of 20 mg/L.
• the cooled medium was used to pour 8, 100 mm x 15 mm petri dishes.
• bacteria were resuspended in filter-sterilized Schenk and Hildebrandt medium containing 0.6 M mannitol and 20 mg/L acetosyringone at pH 5.6 for at least one hour before inoculation.
• filter-sterilized Schenk and Hildebrandt medium containing 0.6 M mannitol and 20 mg/L acetosyringone at pH 5.6 for at least one hour before inoculation.
• pieces of Type I callus from the 3 different duckweed strains and from L. gibba G3 (positive control) were dipped, in the bacterial solution for 2-5 minutes.
• callus pieces were blotted, then transferred as clumps to two plates (for each duckweed strain) of co-cultivation medium, as described above. All callus was incubated at 23°C in the dark for two days.
• Lemna gibba G3 fronds were used to test the effect of three co-cultivation media on rescue of fronds expressing GUS and growing on kanamycin selection medium.
• Fronds were grown for 3 days on liquid Schenk and Hildebrandt medium containing 1% sucrose, and 10 ⁇ M indoleacetic acid prior to use.
• the bacterial strain, AT656, was grown overnight on potato dextrose agar containing 20 mg/L acetosyringone and 50 mg/L kanamycin sulfate at 28°C.
• Three solid media were used for co-cultivation: 1) Schenk and Hildebrandt medium (SH) containing 1% sucrose, 1% agar, 20 mg/L acetosyringone, and 10 ⁇ M indoleacetic acid, 2) Murashige and Skoog medium (MS) containing 3% sucrose, 1% agar, 20 mg/L acetosyringone, and 50 ⁇ M 2,4-dichlorophenoxyacetic acid (2,4-D), and 3) Murashige and Skoog medium containing 3% sucrose, 1% agar, 20 mg/L acetosyringone, 5 ⁇ M 2,4-D, 10 ⁇ M naphthaleneacetic acid, 10 ⁇ M giberrellic acid G3, and 2 ⁇ M benzyladenine.
• SH Schenk and Hildebrandt medium
• MS Murashige and Skoog medium
• MS Murashige and Skoog medium containing 3% sucrose, 1% agar
• the media were prepared, the pH adjusted to 5.6 (SH) or 5.8 (both MS types), autoclaved, cooled, heat labile components acetosyringone, indoleacetic acid and giberrellic acid added as filter-sterilized solutions, and the medium poured into 100 mm x 15 mm petri dishes. For each medium, 20 petri dishes (500 ml) were prepared.
• the fronds from 3 plates were transferred into 3 flasks with 25 ml of liquid media and were grown under 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec.
• the fronds on 15 plates of solid media were divided into 2 groups: 1) the fronds from 10 original plates were transferred to 12 new plates and incubated in the dark (12 plates), 2) the fronds from 5 original plates were transferred to 6 new plates and incubated under subdued light conditions of less than 5 ⁇ mol/m 2 . sec.
• kanamycin sulfate at either 10 mg/L (about 25% of the tissue) or 2 mg/L (about 75% of the remaining tissue) was included.
• a subsample of tissue from both kanamycin treatments was stained for GUS expression.
• Three types of staining was present: 1) staining associated with the original, co-cultivated fronds, 2) staining associated with Type I callus, and 3) staining associated with Type III callus.
• the frequency of callus staining was not high, estimated at about 5-8 fronds giving rise to a kanamycin resistant culture per hundred fronds co-cultivated. Incubation and subculturing of the tissue was continued for another 5 weeks under subdued light.
• tissue remaining was from cultures on MS medium containing 2,4-D, NAA, GA3 and BA.
• the tissue was transferred to Murashige and Skoog medium with 1 ⁇ M 2,4-D, 2 ⁇ M BA, 0.15 g L Gelrite, 0.4 g/L Difco Bacto- agar, 500 mg/L timentin and 10 mg/L kanamycin sulfate.
• Heat labile components were filter- sterilized and added to autoclaved, cooled medium. Healthy tissue that had proliferated from each originally co-cultivated frond was transferred to an individual petri dish.
• kanamycin resistant callus lines were established. These compact Type I callus and Type III callus cultures were characterized by growth on 10 mg/L kanamycin in the light. Eight kanamycin resistant callus cultures were proliferated from 360 original co-cultivated fronds. As these eight lines developed, subsamples of the callus were transferred to half-strength Schenk and Hildebrandt medium containing 0.5% sucrose to regenerate fronds. Of these eight, three regenerated fronds in the absence of kanamycin, frond regeneration would not occur in the presence of kanamycin. None of these fronds showed GUS expression when stained.
• Type I callus was tested for its ability to give GUS expression and kanamycin sulfate resistant cultures following co-cultivation in the presence of Agrobacterium strain C58sZ707pBI121.
• Type I callus was produced by growing Lemna gibba G3 fronds on solid Murashige and Skoog medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 5 ⁇ M 2,4-D, and 2 ⁇ M BA. Callus induction and all subsequent culture was at 23 °C and under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec. After 4 weeks of callus induction, Type I callus clumps were separately cultured on the same medium with the 2,4-D concentration reduced to 1 ⁇ M. The callus was subcultured to fresh medium every two weeks until sufficient callus was proliferated for experimentation.
• Agrobacterium strain C58sZ707pBI121 was streaked on potato dextrose agar with 20 mg/L acetosyringone, 500 mg/L streptomycin, 50 mg/L spectinomycin, and 50 mg/L kanamycin sulfate and grown overnight at 28°C.
• the bacteria were resuspended in filter-sterilized Schenk and Hildebrandt medium containing 0.6M mannitol and 20 mg/L acetosyringone at pH 5.6 for at least one hour before use. For inoculation.
• Type I callus pieces were placed in bacterial solution. For co-cultivation, the pieces were blotted then transferred to MS co-cultivation medium described above. All callus pieces were co-cultivated for two days at 23°C in the dark. After co-cultivation, a subsample of callus pieces were histochemically stained for GUS expression. The results showed a high frequency of GUS expression of varying intensity.
• the approximately 200 remaining callus pieces were transferred to decontamination medium.
• 500 ml of solid MS medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 1 ⁇ M 2,4-D, and 2 ⁇ M BA was prepared, the pH adjusted to 5.6, autoclaved for 20 minutes at 121°C , and cooled.
• a filter-sterilized solution containing cefotaxime was added to the cooled medium to a final medium concentration of 500 mg/L.
• the cooled medium was used to pour 20 plates.
• Approximately 10 callus pieces, each, were transferred to the 20 petri dishes of decontamination medium. All petri dishes were incubated at 23°C in the dark. Weekly subcultures of the callus pieces to identical fresh medium were done and the callus was incubated under the same conditions.
• a small subsample of callus tissue was stained for GUS expression. Expression was present at high frequency and at varying intensity.
• the remaining callus pieces were transferred to selection medium.
• 500 ml of MS with 3% sucrose, 0.15% Gelrite and 0.4% Difco Bacto- agar, supplemented with 1 ⁇ M 2,4-D and 2 ⁇ M BA was prepared, the pH adjusted to 5.6, autoclaved for 20 minutes at 121°C , and cooled.
• a filter-sterilized solution containing cefotaxime, carbenicillin, and kanamycin sulfate was added to the cooled medium to a final medium concentration of 500, 500 and 2 mg/L, respectively.
• the cooled medium was used to pour 20 plates. Approximately 9-10 callus pieces were transferred to the 20 petri dishes of selection medium.
• the callus was incubated at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec.
• Callus culture on water agar was continued for six weeks, with subculture to fresh water agar at two-week intervals.
• week six the callus from all lines had turned yellowish and brown.
• the callus was transferred at the end of week 6 to either solid or liquid, half-strength Schenk and Hildebrandt medium containing 0.5% sucrose and 0.8% Difco Bacto-agar (solid medium only). After 4-6 weeks the callus had organized green nodules that differentiated into thickened, frond like structures.
• fronds could be detached from the callus clumps they were transferred to full-strength Schenk and Hildebrandt medium containing 1 % sucrose, with incubation at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m • sec. These fronds proliferated in liquid SH medium indefinitely. The fronds proliferated equally well on SH medium with or without kanamycin. Bleaching of fronds was not seen in the presence of kanamycin. Frond subsamples were taken periodically and stained for GUS expression. All fronds showed GUS expression.
• the 12.8 kb plasmid DNA was digested with restriction enzymes EcoRl and Hind III to produce a 3.2 kb fragment consisting of the ⁇ -glucuronidase gene and an approximately 9 kb fragment containing the neomycin phosphotransferase gene. Both fragments were isolated from the agarose gel and radioactively labeled by random priming using the Prime-a-Gene kit (Promega). Using these probes, hybridization was done with blots carrying untransformed duckweed DNA and either DNA from transformed line A or transformed line D. li The hybridization reaction was carried out at 65°C overnight in a hybridization oven. The membrane was washed under stringent conditions of 0.1X SSC, 0.1% SDS.
• Type I callus was tested for its ability to give GUS expression and kanamycin sulfate resistant cultures following co-cultivation in the presence of Agrobacterium strain C58sZ707pBI 121.
• Type I callus was produced by growing Lemna gibba G3 fronds on solid Murashige and Skoog medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 5 ⁇ M 2,4-D, and 2 ⁇ M BA. Callus induction and all subsequent culture was at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m 2 • sec. After 4 weeks of callus induction, Type I callus clumps were separately cultured on the same medium with the 2,4-D concentration reduced to 1 ⁇ M. The callus was subcultured to fresh medium every two weeks until sufficient callus was proliferated for experimentation.
• 750 ml of solid Murashige and Skoog medium (MS) with 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 1 ⁇ M 2,4-D and 2 ⁇ M BA was prepared, the pH adjusted to 5.6, autoclaved at 121°C for 20 minutes, and cooled.
• a filter- sterilized solution of acetosyringone was added to the cooled medium to a final concentration of 20 mg/L.
• the cooled medium was used to pour 30, 100 mm x 15 mm petri dishes.
• Agrobacterium strain C58sZ707pBI121 was streaked on potato dextrose agar with 20 mg/L acetosyrin Vgofne, 500 mg/L streptomycin, 50 mg/L spectinomycin, and 50 mg/L kanamycin sulfate and grown overnight at 28°C.
• a randomized block experimental design with one bacterial strain treatment with one replication with 30 petri dishes per replication and approximately 5 callus pieces per petri dish was used.
• the bacteria were resuspended in filter-sterilized MS medium containing 0.6M mannitol and 20 mg/L acetosyringone at pH 5.8 for at least one hour before use.
• Type I callus pieces were placed in bacterial solution.
• co-cultivation the pieces were blotted then transferred to MS co-cultivation medium described above. All callus pieces were co- cultivated for two days at 23°C in the dark. After co-cultivation, a subsample of callus pieces were histochemically stained for GUS expression. The results showed a high frequency of GUS expression.
• the approximately 150 remaining callus pieces were transferred to decontamination medium.
• decontamination medium 750 ml of solid MS medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 1 ⁇ M 2,4-D, and 2 ⁇ M BA was prepared, the pH adjusted to 5.8, autoclaved for 20 minutes at 121°C, and cooled.
• a filter-sterilized solution containing cefotaxime and carbenicillin was added to the cooled medium to a final medium concentrations of 500 mg/L, each.
• the cooled medium was used to pour 30 plates. Approximately 5 callus pieces were transferred to each of the 30 petri dishes of decontamination medium. The callus was then incubated at 23°C in the dark. Weekly subcultures of the callus pieces to identical fresh medium were done and the callus was incubated under the same conditions.
• kanamycin resistant callus lines were begun on week 5. For selection, 750 ml of solid MS medium containing 3% sucrose, 0.15% Gelrite, 0.4% Difco Bacto-agar, 1 ⁇ M 2,4-D, and 2 ⁇ M BA was prepared, the pH adjusted to 5.8, autoclaved for 20 minutes at 121°C, and cooled. A filter-sterilized solution containing cefotaxime, carbenicillin, and kanamycin was added to a final medium concentration of 500 mg/L, 500 mg/L, and 2 mg/L, respectively. The cooled medium was used to pour 30 plates. Approximately 5 callus pieces were transferred to each of the 30 petri dishes of selection medium. The callus was then incubated at 23°C in the dark.
• the light intensity was increased to 40 ⁇ mol/m • sec.
• the callus was maintained on a two-week subculture regime on identical medium and incubation conditions.
• Fronds were regenerated following the procedure of Example 42. Frond proliferation was normal when grown in the presence or absence of kanamycin sulfate, and no bleaching of the fronds was observed. Fronds also showed intense
• the following method is preferred for transforming duckweed callus with Agrobacterium, followed by selection and regeneration of transformed plants.
• Lemna minor has a particularly vigorous callus system, which makes it easier to regenerate transformed plants from this species.
• callus transformation, selection, and frond regeneration is dependent upon a well-established callus system and a number of parameters optimized for each step of the process.
• a vigorously growing callus culture is maintained as described in Example 16.
• Agrobacteria are grown (on Potato Dextrose Agar with appropriate antibiotics and 100 ⁇ M acetosyringone) and resuspended as in Example 32, except that the preferred resuspension medium is MS rather than SH.
• Callus pieces are inoculated by immersing in the solution of resuspended bacteria for a minimum of 3-5 minutes, blotted to remove excess fluid, and plated on co- cultivation medium consisting of MS supplemented with auxin and cytokinin optimized to promote callus growth and 100 ⁇ M acetosyringone. Inoculated callus is incubated in darkness for 2 days.
• callus is transferred to fresh media containing antibiotics to decontaminate the cultures from infecting Agrobacteria.
• the preferred medium is MS with 3% sucrose, 1 ⁇ M 2,4-D, 2 ⁇ M BA, gelled with 0.15% Gelrite and 0.4% Difco Bacto-agar and antibiotic(s).
• the callus is incubated under subdued light of 3-5 ⁇ mol/m 2 . sec.
• the callus is transferred every 2-5 days, 3 days is preferred, to fresh medium of the same composition. The total recovery period lasts for 2-3 weeks, 3-6 subcultures.
• Callus selection follows after the recovery period. Callus is transferred to MS medium supplemented with 1 ⁇ M 2,4-D, 2 ⁇ M BA, 3% sucrose, 0.4% Difco Bacto- agar, 0.15% Gelrite, and 10 mg/L kanamycin sulfate. The callus is incubated under subdued light of 3-5 ⁇ mol/m 2 . sec, with transfer to fresh medium of the same composition every 2 weeks. The callus is maintained in this way for 4-6 weeks. Then the callus is incubated under full light of 40 ⁇ mol/m . sec on the same medium. Selection is considered complete when the callus shows vigorous growth on the selection agent.
• Callus showing vigorous growth on callus maintenance medium in the presence of the selection agent is transferred to regeneration medium to organize and produce plants.
• duckweed regenerates on lean media.
• the selection agent is not present in the regeneration medium.
• the callus is incubated, under full light, on regeneration medium for 2-4 weeks until fronds appear. Fully organized fronds are transferred to liquid S tH? medium with 1-3% sucrose and no plant growth regulators and incubated under full light for further clonal proliferation.
• Example 45 The effect of light intensity and kanamycin sulfate concentration were tested for its effect on the frequency of transformation of Lemna minor callus cultures.
• Lemna minor fronds were grown in liquid Schenk and Hildebrandt medium containing 1% sucrose for two weeks at 23°C under a 16 hr light/8 hr dark photoperiod with light intensity of approximately 40 ⁇ mol/m .sec prior to callus induction. Callus induction was accomplished as in Example 14 using fronds from Lemna minor strain 8744. Callus was maintained on MS medium containing 3% sucrose, 1 ⁇ M 2,4-D, 2 ⁇ M BA, 0.4% Bacto-agar and 0.15% Gelrite for 13 weeks prior to co-cultivation. Callus was subcultured to fresh medium every 2 weeks during this 13-week period.
• solid MS medium with 3% sucrose, 1 ⁇ M 2,4-D, 2 ⁇ M BA, 0.4% Bacto-Agar, and 0.15% Gelrite was prepared, the pH was adjusted to 5.6, the medium was autoclaved at 121°C for 20 minutes, and cooled. A filter-sterilized solution of acetosyringone was added to the cooled medium to a final concentration of 100 ⁇ M. The cooled medium was used to pour 8, 100 mm x 15 mm petri dishes.
• the Agrobacteria were resuspended in filter-sterilized, MS medium containing 0.6 M mannitol and 100 ⁇ M acetosyringone at pH 5.6 for at least one hour before inoculation.
• MS medium containing 0.6 M mannitol and 100 ⁇ M acetosyringone at pH 5.6 for at least one hour before inoculation.
• approximately 160 pieces of Type I callus were dipped in the bacterial solution for 2-5 minutes in batches of 20 callus pieces.
• callus pieces were blotted, then transferred as clumps to co-cultivation medium, 20 callus clumps per 100 mm x 15 mm petri dish. All inoculated callus was incubated at 23°C in the dark for 2 days.
• callus was subcultured to fresh, antibiotic-containing medium every week.
• half (40 callus clumps) of the callus from each light treatment was transferred to fresh medium in which the kanamycin concentration was increased from 10 mg/L to 40 mg/L.
• the remaining 40 callus clumps were transferred to fresh medium maintaining the original kanamycin concentration of 10 mg/L.
• Frond regeneration medium consisted of half-strength Schenk and Hildebrandt medium containing 1% sucrose, 0.4% Bacto- agar, and 0.15% Gelrite. Callus clumps were transferred to fresh medium of the same composition every 2 weeks. Fronds regenerated from callus clumps 3-6 weeks after transfer to regeneration medium.
• Lemna minor genotype of the frequency of rescue of transformed fronds was tested using Lemna minor callus cultures from strain 8627.
• kanamycin selection following co-cultivation, 180 callus clumps were transferred to MS medium containing 1 ⁇ M 2,4-D, 2 ⁇ M BA, 500 mg/L carbenicillin, 500 mg/L cefotaxime and 10 mg/L kanamycin sulfate. All callus was incubated under subdued light levels of less than 5 ⁇ mol/m .sec. On the second week after inoculation, half the callus pieces were transferred to fresh selection medium in which the kanamycin sulfate concentration was increased from 10 mg/L to 40 mg/L, the rest were transferred to fresh selection medium containing 10 mg/L of kanamycin sulfate. Weekly subculture was continued through week 5, post-inoculation at which time subcultures were done every two weeks.
• Example 47 The effect of medium composition on frond regeneration from L. minor callus cultures was also tested. Seven media formulations were tested: (1) water agar, (2) water agar with 100 ⁇ M adenine sulfate, (3) water agar with 10 ⁇ M BA, (4) water agar with 10 ⁇ M BA and 1 ⁇ M IBA, (5) half-strength SH, (6) half-strength SH with 10 ⁇ M BA, and (7) half-strength SH with 11°0 ⁇ M BA and 1 ⁇ M IBA. Callus cultures from both strain 8744 and strain 8627 proliferated in a previous callus induction medium as in Example 12 were used for this experiment. Callus was incubated on the seven different media for 8 weeks, with continual observation for the development of fronds.
• the efficiency of the duckweed system for mammalian gene expression was tested using a human ⁇ -hemoglobin gene construct and a P450 oxidase construct.
• Two Agrobacterium strains were used to inoculate Type I callus of Lemna minor strain 8627.
• strain C58 CI harboring 3 plasmids: pGV3850, pTVK291, pSLD34 was used.
• pTVK291 contains the supervirulence G gene from pTiBo542.
• pSLD34 is an Agrobacterium binary plasmid, derived from pBIN19, consisting of a neomycin phosphotransferase gene under the control of CaMV35S promoter, and a human ⁇ -hemoglobin gene driven by the super- mac promoter.
• strain C58 CI harboring 3 plasmids: pGV3850, pTVK291 and pSLD35 were used.
• the T-DNA is carried on the binary plasmid, pSLD35, which is similar in structure to pSLD34, with the exception that pS:D35 does not contain the ⁇ -hemoglobin gene and instead contains DNA sequences encoding 3 proteins: a human P450 oxidase, an oxidoreductase, and a cytochrome B5. Each gene is driven by a super-mac promoter.
• the pSLD35 plasmid contains both hygromycin and kanamycin selectable marker genes.
• callus clumps were transferred to MS medium containing 1 ⁇ M 2,4-D, 2 ⁇ M BA, 500 mg/L carbenicillin, 500 mg/L cefotaxime and two concentrations of kanamycin: 10 mg/L and 40 mg/L.
• the callus cultures were further divided during incubation with half of the callus pieces on each kanamycin concentration going to subdued light and the other half being incubated under full light.
• Callus was subcultured to fresh medium of the same composition at weekly intervals for the first four weeks after co-cultivation. At week 5, all cultures were incubated under full light intensity for another 6 weeks, with subculture to fresh medium every two weeks.
• Frond regeneration was accomplished using the appropriate media for frond regeneration from L. gibba G3 or L. minor strains as described in Example 42 and Example 47. Fronds regenerated after 3-4 weeks on regeneration medium. Regenerated fronds were maintained on SH medium with 1% sucrose.

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